RESUMO
Detection of self-reactive antibodies has an established role in the diagnosis and monitoring of many human autoimmune diseases. Autoantibodies with restricted reactivity to cytoplasmic compartments and structures are an occasional incidental finding following routine examination of serum for antinuclear antibody reactivity. A prerequisite for rational exploitation of self-reactive antibodies, in either clinical or research settings, is the establishment of the molecular identity of the target autoantigen(s). Here we report on the identification of a novel autoantigen that co-localizes with a subset of cytoplasmic microbodies marked by ABCD3 (PMP-70) and/or PXF (PEX19). Immunoscreening a HeLa cell cDNA expression library with a human autoimmune serum identified two clones that encode fragments of limkain b1 (LKAP). We demonstrate that mouse polyclonal antibodies raised against a bacterially expressed fragment of limkain b1 mark the same cytoplasmic structures as human serum, as does an EGFP:LKAPCT429 fusion protein expressed in HeLa cells. An immunoblot screen against a bacterially expressed MBP:LKAPCT429 fusion protein substrate, using a cohort of 16 additional human sera that display Hep 2 cell cytoplasmic staining patterns similar to the prototype serum, identified three additional sera reactive to limkain b1. This is the first report establishing the molecular identity of a peroxisomal autoantigen. Preliminary results suggest that limkain b1 may be a relatively common target of human autoantibodies reactive to cytoplasmic vesicle-like structures.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Autoantígenos/análise , Proteínas de Membrana/genética , Peroxissomos/imunologia , Transportadores de Cassetes de Ligação de ATP/imunologia , Idoso , Anticorpos Anticitoplasma de Neutrófilos/genética , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoanticorpos/genética , Autoanticorpos/imunologia , Autoantígenos/genética , Autoantígenos/imunologia , Proteínas de Ciclo Celular , Células Cultivadas , DNA Circular/genética , DNA Circular/imunologia , Endorribonucleases , Técnica Indireta de Fluorescência para Anticorpo/métodos , Biblioteca Gênica , Células HeLa , Humanos , Masculino , Proteínas de Membrana/imunologia , Peroxissomos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Transfecção/métodos , Transgenes/genéticaRESUMO
Summary The ratio of osteoprotegerin [OPG, tumour necrosis factor receptor superfamily, member 11b (TNFRSF11B)] to receptor activator of nuclear factor kappaB ligand [RANKL, tumour necrosis factor (ligand) superfamily, member 11 (TNFSF11)] in bone is critical for the regulation of bone remodelling. Myeloma cells can home to bone, triggering increased RANKL and decreased OPG expression by stromal cells, leading to osteolysis. Whether myeloma cells contribute directly to the pool of RANKL or OPG in bone has been contentious. Here we provide evidence of RANKL expression by reverse transcription polymerase chain reaction and in situ hybridization, demonstrating transcripts encoding both the membrane-bound and secreted forms of RANKL in five human multiple myeloma cell lines (LP-1, NCI-H929, OPM-2, RPMI8226, U266) and myeloma cells purified from bone marrow aspirates of myeloma patients. We demonstrated that RANKL encoding mRNAs are translated to protein by antibody detection of RANKL. In vitro assays showed that myeloma cells induced bone marrow derived mononuclear cells to differentiate into adherent tartrate-resistant acid phosphatase positive multinucleated cells, indicative of the formation of functional osteoclasts. This differentiation could also be achieved with passaged myeloma media alone, implicating secreted products. Finally, we provide evidence that the differentiation observed is at least in part the result of myeloma cell expression of RANKL. We therefore conclude that myeloma cells can directly contribute to the pool of RANKL in bone.
