We analyzed the heterogeneity of Drosophila hemocytes on the basis of the expression of cell-type specific antigens. The antigens characterize distinct subsets which partially overlap with those defined by morphological criteria. On the basis of the expression or the lack of expression of blood cell antigens the following hemocyte populations have been defined: crystal cells, plasmatocytes, lamellocytes and precursor cells. The expression of the antigens and thus the different cell types are developmentally regulated. The hemocytes are arranged in four main compartments: the circulating blood cells, the sessile tissue, the lymph glands and the posterior hematopoietic tissue. Each hemocyte compartment has a specific and characteristic composition of the various cell types. The described markers represent the first successful attempt to define hemocyte lineages by immunological markers in Drosophila and help to define morphologically, functionally, spatially and developmentally distinct subsets of hemocytes.

BackgroundPemphigoids are rare diseases associated with IgG, IgE and IgA autoantibodies against collagen XVII/BP180. An entity of the pemphigoid group is the lamina lucida-type of linear IgA disease (IgA pemphigoid) characterized by IgA autoantibodies against BP180. While for the detection of IgG and IgE autoantibodies specific to collagen XVII several ELISA systems have been established, no quantitative immunoassay has been yet developed for IgA autoantibodies. Therefore, the aim of the present study was to develop an ELISA to detect IgA autoantibodies against collagen XVII in the sera of patients with pemphigoids.MethodsWe expressed a soluble recombinant form of the collagen XVII ectodomain in mammalian cells. Reactivity of IgA autoantibodies from patients with IgA pemphigoid was assessed by immunofluorescence microscopy and immunoblot analysis. ELISA test conditions were determined by chessboard titration experiments. The sensitivity, specificity and the cut-off were determined by receiver-operating characteristics analysis.ResultsThe optimized assay was carried out using sera from patients with IgA pemphigoid (n = 30) and healthy donors (n = 105). By receiver operating characteristics (ROC) analysis, an area under the curve of 0.993 was calculated, indicating an excellent discriminatory capacity. Thus, a sensitivity and specificity of 83.3% and 100%, respectively, was determined for a cut-off point of 0.48. As additional control groups, sera from patients with bullous pemphigoid (n = 31) and dermatitis herpetiformis (n = 50), a disease associated with IgA autoantibodies against epidermal transglutaminase, were tested. In 26% of bullous pemphigoid patients, IgA autoantibodies recognized the ectodomain of collagen XVII. One of 50 (2%) of dermatitis herpetiformis patients sera slightly topped the cut-off value.ConclusionsWe developed the first ELISA for the specific and sensitive detection of serum IgA autoantibodies specific to collagen XVII in patients with pemphigoids. This immunoassay should prove a useful tool for clinical and translational research and should essentially improve the diagnosis and disease monitoring of patients with IgA pemphigoid. Moreover, our findings strongly suggest that IgA pemphigoid and IgG bullous pemphigoid represent two ends of the clinical spectrum of an immunological loss of tolerance against components of hemidesmosomes, which is mediated by both IgG and IgA autoantibodies.

The pathomechanism of antibody-mediated tissue damage in autoimmune diseases can be best studied in experimental models by passively transferring specific autoantibodies into animals. The reproduction of the disease in animals depends on several factors, including the cross-reactivity of patient autoantibodies with the animal tissue. Here, we show that autoantibodies from patients with epidermolysis bullosa acquisita (EBA), a subepidermal autoimmune blistering disease, recognize multiple epitopes on murine collagen VII. Indirect immunofluorescence microscopy revealed that EBA patients' IgG cross-reacts with mouse skin. Overlapping, recombinant fragments of murine collagen VII were used to characterize the reactivity of EBA sera and to map the epitopes on the murine antigen by ELISA and immunoblotting. The patients' autoantibody binding to murine collagen VII triggered pathogenic events as demonstrated by a complement fixing and an ex vivo granulocyte-dependent dermal-epidermal separation assay. These findings should greatly facilitate the development of improved disease models and novel therapeutic strategies.

Previous infection with Epstein-Barr virus (EBV) is believed to trigger autoimmunity and to drive autoantibody generation as occurring in patients with systemic lupus erythematosus (SLE). Complement C1q and autoantibodies targeting it (anti-C1q) are also considered to be involved in the pathogenesis of SLE, independently of the impact of environmental insults. Still, the circumstances under which these autoantibodies arise remain elusive. By studying a major antigenic site of C1q targeted by anti-C1q (A08), we aimed to determine environmental factors and possible mechanisms leading to the development of anti-C1q. First, we determined antigenic residues of A08 that were critical for the binding of anti-C1q; importantly, we found the binding to depend on amino-acid-identity. Anti-C1q of SLE patients targeting these critical antigenic residues specifically cross-reacted with the EBV-related EBNA-1 (Epstein-Barr virus nuclear antigen 1)-derived peptide EBNA348. In a cohort of 180 SLE patients we confirmed that patients that were seropositive for EBV and recognized the EBNA348 peptide had increased levels of anti-A08 and anti-C1q, respectively. The correlation of anti-EBNA348 with anti-A08 levels was stronger in SLE patients than in matched healthy controls. Finally, EBNA348 peptide-immunization of C1q−/− mice induced the generation of cross-reactive antibodies which recognized both the A08 epitope of C1q and intact C1q. These findings suggest that anti-C1q in SLE patients could be induced by an EBV-derived epitope through molecular mimicry, thereby further supporting the pathogenic role of EBV in the development of SLE. Considering the role of C1q and anti-C1q, modifying the anti-EBV response might be a promising strategy to improve the course of the disease.

The cross-talk between the inflammatory complement system and hemostasis is becoming increasingly recognized. The interaction between complement C1q, initiation molecule of the classical pathway, and von Willebrand factor (vWF), initiator molecule of primary hemostasis, has been shown to induce platelet rolling and adhesion in vitro. As vWF disorders result in prolonged bleeding, a lack of C1q as binding partner for vWF might also lead to an impaired hemostasis. Therefore, this study aimed to investigate the in vivo relevance of C1q-dependent binding of vWF in hemostasis. For this purpose, we analyzed parameters of primary and secondary hemostasis and performed bleeding experiments in wild type (WT) and C1q-deficient (C1qa −/−) mice, with reconstitution experiments of C1q in the latter. Bleeding tendency was examined by quantification of bleeding time and blood loss. First, we found that complete blood counts and plasma vWF levels do not differ between C1qa −/− mice and WT mice. Moreover, platelet aggregation tests indicated that the platelets of both strains of mice are functional. Second, while the prothrombin time was comparable between both groups, the activated partial thromboplastin time was shorter in C1qa −/− mice. In contrast, tail bleeding times of C1qa −/− mice were prolonged accompanied by an increased blood loss. Upon reconstitution of C1qa −/− mice with C1q, parameters of increased bleeding could be reversed. In conclusion, our data indicate that C1q, a molecule of the first-line of immune defense, actively participates in primary hemostasis by promoting arrest of bleeding. This observation might be of relevance for the understanding of thromboembolic complications in inflammatory disorders, where excess of C1q deposition is observed.