Katja Bieber

Generation of antibodies of distinct subclasses and specificity is linked to H2s in an active mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease, characterized by antibodies to type VII collagen (COL7). EBA can be induced in mice by immunization with a fragment of the non-collagenous 1 domain of murine COL7. Contrary to other autoimmune diseases, e.g., rheumatoid arthritis, little is known about the genetic susceptibility for EBA. We therefore used the EBA mouse model to address the hypothesis that disease induction depends on the major histocompatibility complex (MHC) haplotype. Mice from different inbred strains were immunized with recombinant murine COL7. Five distinct responses were observed: induction of (i) severe disease in SJL/J (H2s) and female MRL/MpJ (H2k), (ii) mild and transient disease in C57Bl/10.s (H2s), (iii) microscopic blistering in DBA/1J (H2q), (iv) only presence of non-pathogenic autoantibodies in C57Bl/6J (H2b), NZM2410/J (H2z), BXD2 (H2b), and male MRL/MpJ, and (v) complete resistance to EBA in NOD/ShiLtJ (H2g7) and C57Bl/10.q (H2q) mice. Overall, susceptibility to EBA was strongly associated with H2s. In addition, the diseased phenotype was associated with autoantibodies to specific regions of COL7. Our findings show that induction of antibodies with a distinct specificity is linked to the MHC haplotype in experimental EBA. Furthermore, our data are the basis for future studies with the goal of identifying non-MHC EBA susceptibility genes.

Mechanisms of Autoantibody-Induced Pathology

Autoantibodies are frequently observed in healthy individuals. In a minority of these individuals, they lead to manifestation of autoimmune diseases, such as rheumatoid arthritis or Graves’ disease. Overall, more than 2.5% of the population is affected by autoantibody-driven autoimmune disease. Pathways leading to autoantibody-induced pathology greatly differ among different diseases, and autoantibodies directed against the same antigen, depending on the targeted epitope, can have diverse effects. To foster knowledge in autoantibody-induced pathology and to encourage development of urgently needed novel therapeutic strategies, we here categorized autoantibodies according to their effects. According to our algorithm, autoantibodies can be classified into the following categories: (1) mimic receptor stimulation, (2) blocking of neural transmission, (3) induction of altered signaling, triggering uncontrolled (4) microthrombosis, (5) cell lysis, (6) neutrophil activation, and (7) induction of inflammation. These mechanisms in relation to disease, as well as principles of autoantibody generation and detection, are reviewed herein.

Repetitive Immunization Breaks Tolerance to Type XVII Collagen and Leads to Bullous Pemphigoid in Mice

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal–epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.

The CD40-CD40L Pathway Contributes to the Proinflammatory Function of Intestinal Epithelial Cells in Inflammatory Bowel Disease

In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohns disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohns disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-gamma to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-alpha, which exerted synergistical effects with IFN-gamma. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-gamma pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs.

Epidermolysis Bullosa Acquisita: From Pathophysiology to Novel Therapeutic Options.

Epidermolysis bullosa acquisita (EBA) is a prototypic organ-specific autoimmune disease induced by autoantibodies to type VII collagen causing mucocutaneous blisters. In the inflammatory (bullous pemphigoid-like) EBA variant, autoantibody binding is followed by a lesional inflammatory cell infiltration, and the overall clinical picture may be indistinguishable from that of bullous pemphigoid, the latter being the most common autoimmune bullous disease. The last decade witnessed the development of several mouse models of inflammatory EBA that facilitated the elucidation of the pathogenesis of autoantibody-induced, cell-mediated subepidermal blistering diseases and identified new therapeutic targets for these and possibly other autoantibody-driven disorders.

Complement-Fixing Anti-Type VII Collagen Antibodies Are Induced in Th1-Polarized Lymph Nodes of Epidermolysis Bullosa Acquisita-Susceptible Mice

The environment encountered in secondary lymphoid organs (e.g., lymph nodes) influences the outcome of immune responses. Immunization of mice with type VII collagen, an adhesion protein expressed at the cutaneous basement membrane, induces experimental epidermolysis bullosa acquisita (EBA). In this model, clinical disease is associated with the H2s haplotype of the MHC found in SJL/J mice. Most other strains (e.g., BALB/c, C57BL/6, NZM2410/J) are resistant to clinical disease, despite autoantibody production. Comparison of autoantibody response in EBA-resistant and -susceptible mice showed an IgG2-dominated response in the latter. We hypothesized that EBA susceptibility is due to specific cytokine gene expression in draining lymph nodes (dLN). To challenge this hypothesis, EBA-susceptible (SJL/J) and -resistant (BALB/c, C57BL/6) mice were immunized with type VII collagen, followed by analysis of clinical phenotype, subclasses of circulating and tissue-bound autoantibodies, complement activation, and cytokine gene expression in dLN. Disease manifestation was associated with induction of complement-fixing autoantibodies, confirming previous observations. Furthermore, however, IFN-γ/IL-4 ratio in dLN of EBA-susceptible mice was significantly increased compared with EBA-resistant strains, suggesting a Th1 polarization. Immunization of H2s-congenic C57BL/6 mice (B6.SJL-H2s) led to Th1 polarization in dLN and clinical disease. In addition to their cytokine milieu, EBA-susceptible and -resistant mice also differed regarding the expression of FcγR on peripheral leukocytes, in which a higher FcγRIV expression in SJL/J and B6.SJL-H2s mice, compared with C57BL/6, was associated with skin lesions. In summary, blistering in experimental EBA is regulated by both adaptive (divergent class switch recombination due to polarized cytokine expression) and innate (FcγR expression) immune mechanisms.

B Cells, Dendritic Cells, and Macrophages Are Required To Induce an Autoreactive CD4 Helper T Cell Response in Experimental Epidermolysis Bullosa Acquisita

In autoimmune bullous dermatoses (AIBD), autoantibodies induce blisters on skin or mucous membranes, or both. Mechanisms of continued autoantibody production and blistering have been well characterized using AIBD animal models. Mechanisms leading to the initial autoantibody production, however, have not been investigated in detail. Epidermolysis bullosa acquisita (EBA) is an AIBD associated with autoantibodies to type VII collagen (COL7). The majority of EBA patients’ sera recognize the noncollagenous domain 1, including the von Willebrand factor A–like domain 2 (vWFA2). In experimental EBA induced by immunization with GST-COL7, disease manifestation depended on the genetic background, a Th1 polarization, and the GST-tag. In this model, nude mice neither produced autoantibodies nor blisters. It has remained uncertain which APC and T cell subsets are required for EBA induction. We established a novel EBA model by immunization with vWFA2 fused to intein (lacking the GST-tag). All tested mouse strains developed autoantibodies, but blisters were exclusively observed in mice carrying H2s. In immunized mice, CD4 T cells specific for vWFA2 were detected, and their induction required presence of B cells, dendritic cells, and macrophages. Anti-vWFA2 autoantibodies located at the lamina densa bound to the dermal side of salt-split skin and induced blisters when transferred into healthy mice. Absence of CD8 T cells at time of immunization had no effect, whereas depletion of CD4 T cells during the same time period delayed autoantibody production and blisters. Collectively, we demonstrate the pathogenic relevance of Abs targeting the vWFA2 domain of COL7 and show the requirement of APC-induced CD4 T cells to induce experimental EBA.

Animal models for autoimmune bullous dermatoses

Abstract:  Autoimmune bullous dermatoses are a group of severe diseases, which are clinically characterized by blisters and erosions of skin and/or mucous membranes. In order to investigate the pathogenesis of these potentially life‐threatening diseases and to develop more specific therapeutic approaches, animal models have been developed that aim to reproduce the clinical, histological and immunopathological findings. We here review established and novel animal models of autoimmune skin blistering diseases and discuss their applications and limitations.

Dimethylfumarate Impairs Neutrophil Functions

Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases.

Cyclical expression of L-selectin (CD62L) by recirculating T cells

L-Selectin (CD62L) mediates T-cell entry into lymph nodes. Whether the microenvironment modulates L-selectin expression of T cells during diapedesis and transit is unknown. Therefore, L-selectin expression was determined quantitatively on circulating T cells in blood, lymph nodes and thoracic duct by confocal laser scanning microscopy. We show that in contrast to leukocyte function-associated antigen-1 (CD11a/CD18) and ICAM-1 (CD54), L-selectin expression is cyclically expressed on recirculating T cells. It is reduced to approximately 30% of the blood value during entry across high endothelial venules. Within lymph nodes, CD4(+) T-cell subsets maintain reduced L-selectin expression at a similar level in all compartments (T-cell zone, B-cell zone and medulla). After exit, L-selectin is re-expressed to levels comparable to those of T cells in blood. Apparently, L-selectin levels are not only down-regulated during T-cell activation but also routinely reduced while transmigrating within lymph nodes. L-Selectin down-regulation seems to be ligand independent since it also occurs in the white pulp compartments of the spleen which lack classic L-selectin ligands such as GlyCAM-1 and CD34. In addition, T cells in non-lymphoid organs do not reveal reduced L-selectin levels. Thus, the ability of secondary lymphoid organs to reduce L-selectin expression of T cells prior to activation might be a prerequisite for their characteristic property to induce primary immune responses.