Experimental induction of anti-muscarinic type-3-receptor extracellular loop antibodies by immunization with 4-hydroxy-2-nonenal modified Ro60 and unmodified Ro60.
Biji T Kurien, Devavrat Dave, Martha Tsaliki, Syed M S Quadri, Valerie M Lewis, Robert Hal Scofield
{"title":"Experimental induction of anti-muscarinic type-3-receptor extracellular loop antibodies by immunization with 4-hydroxy-2-nonenal modified Ro60 and unmodified Ro60.","authors":"Biji T Kurien, Devavrat Dave, Martha Tsaliki, Syed M S Quadri, Valerie M Lewis, Robert Hal Scofield","doi":"10.1093/cei/uxae114","DOIUrl":null,"url":null,"abstract":"<p><strong>Objective: </strong>Sjögren's Disease (SjD) subjects have decreased lacrimal/salivary gland function. Studies have proposed that autoantibodies targeting G-protein-coupled muscarinic acetylcholine-type-3-receptor (M3R) are potential clinical markers for SjD. We hypothesized that rabbits/mice immunized with 4-hydroxy-2-nonenal (HNE)-modified/unmodified Ro60 will develop an autoimmunity, specifically a SjD phenotype, thus expressing increased levels of anti-M3R antibodies.</p><p><strong>Methods: </strong>We immunized two rabbits each with 10 mM HNE-modified Ro60/unmodified Ro60 antigen or Ro274-290/Ro413-428/Ro500-517 Ro60 peptides. Two rabbits each were immunized with either M3R second extracellular loop (ECL2) or M3R ECL3 peptide. Finally, five groups of BALB/c mice were immunized as follows-Group-I immunized with Ro60, Groups-II-IV immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively and Group-V-Freund's adjuvant. Serum antibodies to M3R ECL2/ECL3/Ro60/La or Sm were detected by ELISA. Functional assays were also performed.</p><p><strong>Results: </strong>Immunization with HNE-modified Ro60/unmodified Ro60 antigen or Ro274/Ro 413/Ro500 peptides induced a rapid intermolecular epitope spreading to M3R ECL2/ECL3, especially to M3R ECL3 in HNE-Ro immunized rabbits. These animals did not bind to scrambled M3R peptides. Ro60-immunized rabbit IgG inhibited M3R activity in a functional assay. Rabbits immunized with ECL2/ECL3 developed high reactivity to Ro60 but not against Sm/RNP. We found a differential antibody-induction against M3R ECL2 with Group-3 mice developing significant reactivity.</p><p><strong>Conclusion: </strong>Our data show induction of increasing anti-M3R antibodies in rabbits immunized with Ro60/HNE-Ro60 or Ro60 peptides and differential induction of these antibodies in mice immunized with Ro60 modified with increasing HNE. These findings suggest that M3R ECL2/ECL3 are involved in SjD autoimmunity progression.</p>","PeriodicalId":10268,"journal":{"name":"Clinical and experimental immunology","volume":" ","pages":""},"PeriodicalIF":3.4000,"publicationDate":"2025-01-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11748001/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and experimental immunology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/cei/uxae114","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"IMMUNOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Objective: Sjögren's Disease (SjD) subjects have decreased lacrimal/salivary gland function. Studies have proposed that autoantibodies targeting G-protein-coupled muscarinic acetylcholine-type-3-receptor (M3R) are potential clinical markers for SjD. We hypothesized that rabbits/mice immunized with 4-hydroxy-2-nonenal (HNE)-modified/unmodified Ro60 will develop an autoimmunity, specifically a SjD phenotype, thus expressing increased levels of anti-M3R antibodies.
Methods: We immunized two rabbits each with 10 mM HNE-modified Ro60/unmodified Ro60 antigen or Ro274-290/Ro413-428/Ro500-517 Ro60 peptides. Two rabbits each were immunized with either M3R second extracellular loop (ECL2) or M3R ECL3 peptide. Finally, five groups of BALB/c mice were immunized as follows-Group-I immunized with Ro60, Groups-II-IV immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively and Group-V-Freund's adjuvant. Serum antibodies to M3R ECL2/ECL3/Ro60/La or Sm were detected by ELISA. Functional assays were also performed.
Results: Immunization with HNE-modified Ro60/unmodified Ro60 antigen or Ro274/Ro 413/Ro500 peptides induced a rapid intermolecular epitope spreading to M3R ECL2/ECL3, especially to M3R ECL3 in HNE-Ro immunized rabbits. These animals did not bind to scrambled M3R peptides. Ro60-immunized rabbit IgG inhibited M3R activity in a functional assay. Rabbits immunized with ECL2/ECL3 developed high reactivity to Ro60 but not against Sm/RNP. We found a differential antibody-induction against M3R ECL2 with Group-3 mice developing significant reactivity.
Conclusion: Our data show induction of increasing anti-M3R antibodies in rabbits immunized with Ro60/HNE-Ro60 or Ro60 peptides and differential induction of these antibodies in mice immunized with Ro60 modified with increasing HNE. These findings suggest that M3R ECL2/ECL3 are involved in SjD autoimmunity progression.
期刊介绍：
Clinical & Experimental Immunology (established in 1966) is an authoritative international journal publishing high-quality research studies in translational and clinical immunology that have the potential to transform our understanding of the immunopathology of human disease and/or change clinical practice.
The journal is focused on translational and clinical immunology and is among the foremost journals in this field, attracting high-quality papers from across the world. Translation is viewed as a process of applying ideas, insights and discoveries generated through scientific studies to the treatment, prevention or diagnosis of human disease. Clinical immunology has evolved as a field to encompass the application of state-of-the-art technologies such as next-generation sequencing, metagenomics and high-dimensional phenotyping to understand mechanisms that govern the outcomes of clinical trials.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
