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Immune responses to Ro60 and its peptides in mice. I. The nature of the immunogen and endogenous autoantigen determine the specificities of the induced autoantibodies.

Deshmukh US, Lewis JE, Gaskin F, Kannapell CC, Waters ST, Lou YH, Tung KS, Fu SM - J. Exp. Med. (1999)

Bottom Line: With human Ro60316-335 as the immunogen, additional autoantibodies reactive with the Golgi complex were found.These results show that both the nature of the immunogen and the immunogenicity of the related endogenous antigen are important in determining the specificities of the autoantibodies generated.They have significant implications for proposed mechanisms on the generation of complex patterns of autoantibodies to a diverse group of autoantigens in SLE patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Anti-Ro60 autoantibodies are found in a variety of autoimmune disorders including systemic lupus erythematosus (SLE), Sjögren's syndrome, primary biliary cirrhosis, and active hepatitis. They are the most prevalent autoantibodies in normal individuals and in asymptomatic mothers of infants afflicted with neonatal lupus. In the present study, immune responses to recombinant human Ro60 (rhRo60) and recombinant mouse Ro60 (rmRo60) and selected Ro60 peptides in non-SLE-prone mice were investigated. Multiple T and B cell epitopes were identified in Ro60. Immunizations with either xenogeneic or autologous Ro60 induced autoantibodies to a diverse group of autoantigens. In addition to La and Ro52, proteins in the small nuclear ribonucleoprotein (snRNP) particles such as SmA, SmB, SmD, and 70-kD U1-RNP were unexpectedly identified as targeted antigens. In the studies involving synthetic Ro60 peptides, both human and mouse Ro60316-335 peptides, which differ in three amino acids, were found to contain dominant cross-reactive T cell determinants. Immunizations with these peptides induced autoantibodies to Ro60, La, SmD, and 70-kD U1-RNP without autoantibodies to Ro52, SmA, or SmB. With human Ro60316-335 as the immunogen, additional autoantibodies reactive with the Golgi complex were found. In contrast to the immunodominance of both human and mouse Ro60316-335 peptides, the T cell determinant in human Ro60441-465 was dominant, whereas that in the mouse peptide was cryptic. Immunization with human Ro60441-465 induced primarily anti-peptide Abs. Mouse Ro60441-465 failed to induce an antibody response. These results show that both the nature of the immunogen and the immunogenicity of the related endogenous antigen are important in determining the specificities of the autoantibodies generated. They have significant implications for proposed mechanisms on the generation of complex patterns of autoantibodies to a diverse group of autoantigens in SLE patients.

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Induction of antibody diversification in mice immunized with peptide mRo60316–335.  (A) Reactivity of day 30 sera  from four mice with different ribonucleoproteins was tested in  slot blots. All sera were tested at  1:250 dilution. (B) Reactivity of  pooled sera from SJL/J mice immunized with hRo60316–335 peptide (lane 1), JS7A (lane 2), and  CFA (lane 3). (C) Pooled sera  from mice immunized with  mRo60316–335 were absorbed  with the immunogen (hatched  bars) and control peptide JS7A  (cross bars). The reactivities of  the unabsorbed (open bars) and  the absorbed sera were determined in ELISA with either  mRo60316–335 (left) or mRo60  (right) as the target antigen. The  sera absorbed with the immunogen did not react with the immunogen mRo60316–335 while  the reactivity with mRo60 remained. Results are expressed as  mean duplicate OD490nm.
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Figure 15: Induction of antibody diversification in mice immunized with peptide mRo60316–335. (A) Reactivity of day 30 sera from four mice with different ribonucleoproteins was tested in slot blots. All sera were tested at 1:250 dilution. (B) Reactivity of pooled sera from SJL/J mice immunized with hRo60316–335 peptide (lane 1), JS7A (lane 2), and CFA (lane 3). (C) Pooled sera from mice immunized with mRo60316–335 were absorbed with the immunogen (hatched bars) and control peptide JS7A (cross bars). The reactivities of the unabsorbed (open bars) and the absorbed sera were determined in ELISA with either mRo60316–335 (left) or mRo60 (right) as the target antigen. The sera absorbed with the immunogen did not react with the immunogen mRo60316–335 while the reactivity with mRo60 remained. Results are expressed as mean duplicate OD490nm.

Mentions: Immunization of mice with peptide mRo60316–335 resulted in high titers of anti-peptide antibodies, capable of immunoprecipitating native mRo60. Reactivity to La, SmD, and U1RNP associated 70-kD protein and DHFR was observed in slot blots (Fig. 15 A) indicative of intermolecular epitope spreading. The reactivity patterns of the immune sera were similar to those of sera of mice immunized with the human peptide (Figs. 7 and 15 B). With respect to intramolecular spreading, absorption of sera with the immunogen had little effect on the reactivity to Ro60 although almost all reactivity to the peptide was abolished (Fig. 15 C). Absorption with control peptide JS7A had no effect, either on the reactivity with the peptide or with the whole antigen. It is of interest to note that these immune sera did not stain the Golgi complex. Similar results were obtained in an additional experiment.


Immune responses to Ro60 and its peptides in mice. I. The nature of the immunogen and endogenous autoantigen determine the specificities of the induced autoantibodies.

Deshmukh US, Lewis JE, Gaskin F, Kannapell CC, Waters ST, Lou YH, Tung KS, Fu SM - J. Exp. Med. (1999)

Induction of antibody diversification in mice immunized with peptide mRo60316–335.  (A) Reactivity of day 30 sera  from four mice with different ribonucleoproteins was tested in  slot blots. All sera were tested at  1:250 dilution. (B) Reactivity of  pooled sera from SJL/J mice immunized with hRo60316–335 peptide (lane 1), JS7A (lane 2), and  CFA (lane 3). (C) Pooled sera  from mice immunized with  mRo60316–335 were absorbed  with the immunogen (hatched  bars) and control peptide JS7A  (cross bars). The reactivities of  the unabsorbed (open bars) and  the absorbed sera were determined in ELISA with either  mRo60316–335 (left) or mRo60  (right) as the target antigen. The  sera absorbed with the immunogen did not react with the immunogen mRo60316–335 while  the reactivity with mRo60 remained. Results are expressed as  mean duplicate OD490nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192918&req=5

Figure 15: Induction of antibody diversification in mice immunized with peptide mRo60316–335. (A) Reactivity of day 30 sera from four mice with different ribonucleoproteins was tested in slot blots. All sera were tested at 1:250 dilution. (B) Reactivity of pooled sera from SJL/J mice immunized with hRo60316–335 peptide (lane 1), JS7A (lane 2), and CFA (lane 3). (C) Pooled sera from mice immunized with mRo60316–335 were absorbed with the immunogen (hatched bars) and control peptide JS7A (cross bars). The reactivities of the unabsorbed (open bars) and the absorbed sera were determined in ELISA with either mRo60316–335 (left) or mRo60 (right) as the target antigen. The sera absorbed with the immunogen did not react with the immunogen mRo60316–335 while the reactivity with mRo60 remained. Results are expressed as mean duplicate OD490nm.
Mentions: Immunization of mice with peptide mRo60316–335 resulted in high titers of anti-peptide antibodies, capable of immunoprecipitating native mRo60. Reactivity to La, SmD, and U1RNP associated 70-kD protein and DHFR was observed in slot blots (Fig. 15 A) indicative of intermolecular epitope spreading. The reactivity patterns of the immune sera were similar to those of sera of mice immunized with the human peptide (Figs. 7 and 15 B). With respect to intramolecular spreading, absorption of sera with the immunogen had little effect on the reactivity to Ro60 although almost all reactivity to the peptide was abolished (Fig. 15 C). Absorption with control peptide JS7A had no effect, either on the reactivity with the peptide or with the whole antigen. It is of interest to note that these immune sera did not stain the Golgi complex. Similar results were obtained in an additional experiment.

Bottom Line: With human Ro60316-335 as the immunogen, additional autoantibodies reactive with the Golgi complex were found.These results show that both the nature of the immunogen and the immunogenicity of the related endogenous antigen are important in determining the specificities of the autoantibodies generated.They have significant implications for proposed mechanisms on the generation of complex patterns of autoantibodies to a diverse group of autoantigens in SLE patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Anti-Ro60 autoantibodies are found in a variety of autoimmune disorders including systemic lupus erythematosus (SLE), Sjögren's syndrome, primary biliary cirrhosis, and active hepatitis. They are the most prevalent autoantibodies in normal individuals and in asymptomatic mothers of infants afflicted with neonatal lupus. In the present study, immune responses to recombinant human Ro60 (rhRo60) and recombinant mouse Ro60 (rmRo60) and selected Ro60 peptides in non-SLE-prone mice were investigated. Multiple T and B cell epitopes were identified in Ro60. Immunizations with either xenogeneic or autologous Ro60 induced autoantibodies to a diverse group of autoantigens. In addition to La and Ro52, proteins in the small nuclear ribonucleoprotein (snRNP) particles such as SmA, SmB, SmD, and 70-kD U1-RNP were unexpectedly identified as targeted antigens. In the studies involving synthetic Ro60 peptides, both human and mouse Ro60316-335 peptides, which differ in three amino acids, were found to contain dominant cross-reactive T cell determinants. Immunizations with these peptides induced autoantibodies to Ro60, La, SmD, and 70-kD U1-RNP without autoantibodies to Ro52, SmA, or SmB. With human Ro60316-335 as the immunogen, additional autoantibodies reactive with the Golgi complex were found. In contrast to the immunodominance of both human and mouse Ro60316-335 peptides, the T cell determinant in human Ro60441-465 was dominant, whereas that in the mouse peptide was cryptic. Immunization with human Ro60441-465 induced primarily anti-peptide Abs. Mouse Ro60441-465 failed to induce an antibody response. These results show that both the nature of the immunogen and the immunogenicity of the related endogenous antigen are important in determining the specificities of the autoantibodies generated. They have significant implications for proposed mechanisms on the generation of complex patterns of autoantibodies to a diverse group of autoantigens in SLE patients.

Show MeSH
Related in: MedlinePlus