title: Circulating microparticles in systemic Lupus Erythematosus.
abstract: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease presenting with a wide array of clinical manifestations and an elusive pathogenesis. A characteristic feature in SLE is the occurrence of autoantibodies against chromatin, double-stranded DNA, and RNA-binding ribonucleoproteins. Observations of defective clearance of dying cells in SLE combined with the generation and exposure of nuclear autoantigens during apoptosis have led to the hypothesis that improperly cleared apoptotic debris constitutes a source of autoantigens capable of triggering autoimmune disease. In blood, circulating, heterogeneous subcellular microparticles (MPs) are released from cells and platelets constitutively and upon cellular activation or apoptosis. Such MPs may reflect the state of their parental cells and tissues, and could serve as markers of pathology. Particular in SLE MPs may serve as carriers of autoantigens and constituents of immune complexes (ICs).  The purposes of this PhD thesis were to develop and apply qualitative and quantitative methods to characterize circulating MPs with respect to numbers, cellular origins and composition in a large cohort of well-characterized SLE patients compared to healthy and disease controls and to explore associations with clinical, biochemical and serological parameters. The PhD thesis consists of a review and three papers.  In the first paper we show that SLE patients have significantly decreased numbers of annexin V binding MPs and MPs from platelets, leukocytes and endothelial cells using flow cytometry. Two morphologically distinguishable populations of annexin V non-binding MPs were increased in the SLE patients. The annexin V non-binding MPs of most likely cellular origin were associated with the presence of lupus nephritis, markers of increased disease activity and levels of endothelial cell-derived MPs.   In the second paper we present the development of a proteomic method to characterize the protein composition of purified MPs using high-resolution mass spectrometry and establish a set of proteins which may serve as normalizers for MP protein quantitation enabling comparison across samples and studies. We identify a core proteome of more than 330 proteins in MPs from healthy individuals. The method enables an unbiased, comprehensive coverage of all proteins present in MPs irrespectively of the availability and utility of immunological reagents.  In the third paper we use the established flow cytometry and mass spectrometry platforms to show that SLE-MPs carry more surface-bound IgG, IgM and C1q indicating that SLE-MPs could be antigenic targets and constituents of ICs. Additionally, the numbers of MPs carrying IgG are also increased in SLE. The load of IgG on SLE-MPs was associated with markers of complement activation, indicators of disease activity in SLE.   In conclusion, using both antibody-dependent and independent methods we demonstrate that SLE-MPs deviate distinctly from controls and may serve as precursors of ICs associated with complement activation and disease activity. This supports the hypothesis of MPs being directly involved in or reflecting tissue-specific or systemic inflammation in addition to carrying accessible antigens. Accordingly, further characterization of the proteome and functional properties of SLE-MPs seem highly warranted in future studies.
authors.affiliation: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Copenhagen S, Denmark. ctn@ssi.dk
authors.forename: Christoffer Tandrup
authors.initials: CT
authors.lastname: Nielsen
mesh_terms: D017304:Annexin A5 / Q000097:blood; D000974:Antibodies, Antinuclear / Q000097:blood*; D000936:Antigen-Antibody Complex / Q000097:blood; D000954:Antigens, Surface / Q000032:analysis; D017209:Apoptosis / Q000276:immunology; D015551:Autoimmunity; D055252:Cell-Derived Microparticles / Q000276:immunology / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D059647:Gene-Environment Interaction; D020022:Genetic Predisposition to Disease; D006801:Humans; D007155:Immunologic Factors / Q000097:blood; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000209:etiology / Q000276:immunology; D013058:Mass Spectrometry / Q000379:methods; D008854:Microscopy, Electron / Q000379:methods; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D017304:Annexin A5; D000974:Antibodies, Antinuclear; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D007155:Immunologic Factors