Intrinsic B cell defects in NZB and NZW mice contribute to systemic lupus erythematosus in (NZBNZW) F1 mice

Intrinsic B cell defects in NZB and NZW mice contribute to systemic lupus erythematosus in (NZBNZW) F1 mice. of autoreactive Az/Ad-restricted T-cell clones. Possible explanations for this are discussed. INTRODUCTION (NZBNZW)F1 (B/WF1) mice have been studied as an animal model for human systemic lupus erythematosus (SLE) (reviewed in 1C3). At around 6 months of age, these mice start to show elevated serum immunoglobulin G (IgG) anti-DNA antibodies and severe immune-complex glomerulonephritis. Female mice are more susceptible to autoimmune symptoms than are male mice. In addition to the abnormality in both B and T lymphocytes,4C7 classic genetic studies revealed that this heterozygosity at the H-2 complex of the murine major histocompatibility complex (MHC) is usually critically involved in the development of B/WF1 autoimmunity.8,9 Our previous studies demonstrated the existence of mixed haplotype Az/Ad major histocompatibility complex (MHC) class II molecules on B/WF1 spleen cells and the existence of autoreactive and foreign antigen-reactive T-cell clones restricted by such Az/Ad molecules.10 Further studies exhibited that some of these autoreactive T-cell clones induced anti-DNA antibody of IgG class in cell transfer experiments.11 These studies suggested that this recognition of self-peptide(s) in association with Az/Ad class II molecules by pathogenic autoreactive T cells is important for the onset and/or progression of B/WF1 autoimmunity. In an accompanying paper, we examined the characteristics of peptides that bind to Az/Ad class II molecules12 and suggested that charged residues in the Nav1.7-IN-3 peptide sequence affect the binding to the Az/Ad molecules. In this study, we attempted to clarify whether unique amino acid residue(s) in the Az/Ad class II molecules might exist which are crucial for the activation of autoreactive T cells. We generated and analysed a panel of transfectant cell lines that express Rabbit polyclonal to ALOXE3 Az/Ad molecules with single amino acid substitutions at several polymorphic sites around the or chain. Our results suggest that alanine at position 69 of the Ad chain is critical for the activation of autoreactive Az/Ad-restricted Nav1.7-IN-3 T-cell clones. Nav1.7-IN-3 MATERIALS AND METHODS Mice New Zealand Black (NZB, H-2d) and New Zealand White (NZW, H-2z) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). (NZBNZW)F1 (B/WF1) mice were generated by mating female NZB with male NZW mice in our animal breeding facilities. Animals in this study were used in accordance with the Saga Medical School Guidelines for Animal Experimentation. Antigens, peptides and monoclonal antibodies (mAbs) Keyhole limpet haemocyanin (KLH) was purchased from Calbiochem-Behring Corp. (La Jolla, CA). Stock solutions (2 mg/ml) Nav1.7-IN-3 were made in Hanks balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY) exceeded through Millipore filters (045 m) and stored at 4. l-plastin 588C605 peptide (SMARKIGARVYALPEDLV, as expressed by the single letter code of amino acid) that was shown to bind Az/AdMHC class II molecules12 was synthesized using the Multipin Peptide Synthesis System (Chiron Mimotopes, Emeryville, CA) according to the manufacturers protocol. The sequence was confirmed by automated Edman degradation using a pulsed liquid protein sequencer 477A equipped with an on-line phenylthiohydantoin (PTH) amino acid analyser 120A (Applied Biosystem, Foster City, OR), and the purity was more than 95% by reversed phase high pressure liquid chromatography (HPLC) analysis. The origins and specificities of anti-class II mAb hybridoma cell lines 10.2.16 (anti-Ak, cross-react to Az),13 K24C199 (anti-Ad)14 and BW/9 (rat anti-mouse monomorphic I-A mAb)15 are described in each reference. Culture supernatants of.

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