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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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Metal ions and topoisomerase I are concentrated in punctate intranucleolar structures. (A)  HeLa cells were fixed in 1% lead acetate, and subsequently stained with  1% ammonium sulfide. Bright field  microscopy demonstrates intense  staining of punctate intranucleolar  structures (nucleolini). (B) HeLa  cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti– human IgG, and were examined by  confocal fluoresence microscopy.  Diffuse nuclear staining, as well as  punctate intranucleolar structures  (nucleolini), are seen. Similar results  were obtained with six other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed  using sera obtained from healthy individuals, or from Ro/La-positive  lupus patients. Bar, 10 μM.
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Figure 5: Metal ions and topoisomerase I are concentrated in punctate intranucleolar structures. (A) HeLa cells were fixed in 1% lead acetate, and subsequently stained with 1% ammonium sulfide. Bright field microscopy demonstrates intense staining of punctate intranucleolar structures (nucleolini). (B) HeLa cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti– human IgG, and were examined by confocal fluoresence microscopy. Diffuse nuclear staining, as well as punctate intranucleolar structures (nucleolini), are seen. Similar results were obtained with six other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed using sera obtained from healthy individuals, or from Ro/La-positive lupus patients. Bar, 10 μM.

Mentions: The most likely subcellular site in which these increased metal concentrations occur is the nucleolus, since the scleroderma autoantigens are enriched in nucleoli (reviewed in reference 33), and several histochemical studies have demonstrated that the nucleolus has the unusual capacity to concentrate metal ions, including silver, zinc, cobalt, and lead (38–41). Morphologic studies were performed to evaluate whether the patterns of staining of scleroderma autoantigens and metals in the nucleolus were similar (Fig. 5). Exclusive staining of punctate intranucleolar structures (nucleolini) was observed when HeLa cells were fixed with lead acetate and stained with ammonium sulfide (Fig. 5 A), or when ethanol-fixed HeLa cells were incubated with a solution containing zinc ions, before staining with dithizone (40, and data not shown). Identical punctate intranucleolar structures were observed when confocal immunofluoresence microscopy was performed with antibodies to topoisomerase I (Fig. 5 B) and other scleroderma autoantigens (data not shown).


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

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

Metal ions and topoisomerase I are concentrated in punctate intranucleolar structures. (A)  HeLa cells were fixed in 1% lead acetate, and subsequently stained with  1% ammonium sulfide. Bright field  microscopy demonstrates intense  staining of punctate intranucleolar  structures (nucleolini). (B) HeLa  cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti– human IgG, and were examined by  confocal fluoresence microscopy.  Diffuse nuclear staining, as well as  punctate intranucleolar structures  (nucleolini), are seen. Similar results  were obtained with six other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed  using sera obtained from healthy individuals, or from Ro/La-positive  lupus patients. Bar, 10 μM.
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Related In: Results  -  Collection

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Figure 5: Metal ions and topoisomerase I are concentrated in punctate intranucleolar structures. (A) HeLa cells were fixed in 1% lead acetate, and subsequently stained with 1% ammonium sulfide. Bright field microscopy demonstrates intense staining of punctate intranucleolar structures (nucleolini). (B) HeLa cells were stained with a monospecific human serum recognizing topoisomerase I and FITC-goat anti– human IgG, and were examined by confocal fluoresence microscopy. Diffuse nuclear staining, as well as punctate intranucleolar structures (nucleolini), are seen. Similar results were obtained with six other monospecific topoisomerase I sera. Nucleolini were never stained when similar experiments were performed using sera obtained from healthy individuals, or from Ro/La-positive lupus patients. Bar, 10 μM.
Mentions: The most likely subcellular site in which these increased metal concentrations occur is the nucleolus, since the scleroderma autoantigens are enriched in nucleoli (reviewed in reference 33), and several histochemical studies have demonstrated that the nucleolus has the unusual capacity to concentrate metal ions, including silver, zinc, cobalt, and lead (38–41). Morphologic studies were performed to evaluate whether the patterns of staining of scleroderma autoantigens and metals in the nucleolus were similar (Fig. 5). Exclusive staining of punctate intranucleolar structures (nucleolini) was observed when HeLa cells were fixed with lead acetate and stained with ammonium sulfide (Fig. 5 A), or when ethanol-fixed HeLa cells were incubated with a solution containing zinc ions, before staining with dithizone (40, and data not shown). Identical punctate intranucleolar structures were observed when confocal immunofluoresence microscopy was performed with antibodies to topoisomerase I (Fig. 5 B) and other scleroderma autoantigens (data not shown).

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