Stefanie Kuerten

Identification of a B cell-dependent subpopulation of multiple sclerosis by measurements of brain-reactive B cells in the blood

B cells are increasingly coming into play in the pathogenesis of multiple sclerosis (MS). Here, we screened peripheral blood mononuclear cells (PBMC) from patients with clinically isolated syndrome (CIS), MS, other non-inflammatory neurological, inflammatory neurological or autoimmune diseases, and healthy donors for their B cell reactivity to CNS antigen using the enzyme-linked immunospot technique (ELISPOT) after 96 h of polyclonal stimulation. Our data show that nine of 15 patients with CIS (60.0%) and 53 of 67 patients with definite MS (79.1%) displayed CNS-reactive B cells, compared to none of the control donors. The presence of CNS-reactive B cells in the blood of the majority of patients with MS or at risk to develop MS along with their absence in control subjects suggests that they might be indicative of a B cell-dependent subpopulation of the disease.

Differential aspects of immune cell infiltration and neurodegeneration in acute and relapse experimental autoimmune encephalomyelitis.

Proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a well-established animal model for the study of relapsing-remitting multiple sclerosis (MS). Here we investigated histomorphological differences between acute and relapse EAE in order to get further insights into the mechanisms that trigger the transition from a relapsing-remitting course to chronic-progressive disease. We found the extent of inflammation to differ in respect to the type of immune cells infiltrating the CNS and the manifestation of edema. Myelin pathology was predominated by demyelinated axons in the acute phase. In relapse EAE the extent of myelin pathology declined and was characterized by a balance between demyelinated and demyelinating nerve fibers. Axonal pathology increased with disease progression and was partly separated from myelin pathology and inflammation. The reported incisive differences between acute and relapse EAE suggest a transition from inflammatory processes to independent neurodegeneration in the disease course.

B1 cells are unaffected by immune modulatory treatment in remitting–relapsing multiple sclerosis patients

In this study we aimed to investigate whether treatment with an immune modulatory drug had an effect on the distribution of B cell subpopulations in patients with remitting-relapsing multiple sclerosis (RRMS). We investigated the first-line drugs glatiramer acetate, interferon-β and natalizumab. Our data show that the frequency of the CD27(+)CD43(+) B1 cell subset was significantly diminished in RRMS patients compared to healthy subjects and that this subset was unaffected by treatment. Regardless of their true nature, we believe that these cells are part of the autoimmune disease pattern.

Nimodipine fosters remyelination in a mouse model of multiple sclerosis and induces microglia-specific apoptosis

Significance Multiple sclerosis (MS) is the most frequent neurological disease that leads to premature retirement in young adults. Progressive MS currently is not only incurable, but also untreatable. Here we show that the calcium channel antagonist nimodipine significantly attenuated clinical disease and central nervous system degeneration and also fostered remyelination in a mouse model of MS. The effect of nimodipine was microglia specific, inducing apoptosis and decreasing the production of neurotoxic molecules such as nitric oxide and reactive oxygen species both in vitro and in vivo. These results introduce a treatment option for MS and also may have broad therapeutic implications for chronic neuroinflammatory diseases in general. Despite continuous interest in multiple sclerosis (MS) research, there is still a lack of neuroprotective strategies, because the main focus has remained on modulating the immune response. Here we performed in-depth analysis of neurodegeneration in experimental autoimmune encephalomyelitis (EAE) and in in vitro studies regarding the effect of the well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated clinical EAE and spinal cord degeneration and promoted remyelination. Surprisingly, we observed calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell-type specific and irrespective of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS and reactive oxygen species levels in EAE. In addition, increased numbers of Olig2+APC+ oligodendrocytes were detected. Overall, nimodipine application seems to generate a favorable environment for regenerative processes and therefore could be a treatment option for MS, because it combines features of immunomodulation with beneficial effects on neuroregeneration.

Categorization of multiple sclerosis relapse subtypes by B cell profiling in the blood

IntroductionB cells are attracting increasing attention in the pathogenesis of multiple sclerosis (MS). B cell-targeted therapies with monoclonal antibodies or plasmapheresis have been shown to be successful in a subset of patients. Here, patients with either relapsing-remitting (n = 24) or secondary progressive (n = 6) MS presenting with an acute clinical relapse were screened for their B cell reactivity to brain antigens and were re-tested three to nine months later. Enzyme-linked immunospot technique (ELISPOT) was used to identify brain-reactive B cells in peripheral blood mononuclear cells (PBMC) directly ex vivo and after 96 h of polyclonal stimulation. Clinical severity of symptoms was determined using the Expanded Disability Status Scale (EDSS).ResultsNine patients displayed B cells in the blood producing brain-specific antibodies directly ex vivo. Six patients were classified as B cell positive donors only after polyclonal B cell stimulation. In 15 patients a B cell response to brain antigens was absent. Based on the autoreactive B cell response we categorized MS relapses into three different patterns. Patients who displayed brain-reactive B cell responses both directly ex vivo and after polyclonal stimulation (pattern I) were significantly younger than patients in whom only memory B cell responses were detectable or entirely absent (patterns II and III; p = 0.003). In one patient a conversion to a positive B cell response as measured directly ex vivo and subsequently also after polyclonal stimulation was associated with the development of a clinical relapse. The evaluation of the predictive value of a brain antigen-specific B cell response showed that seven of eight patients (87.5%) with a pattern I response encountered a clinical relapse during the observation period of 10 months, compared to two of five patients (40%) with a pattern II and three of 14 patients (21.4%) with a pattern III response (p = 0.0005; hazard ratio 6.08 (95% confidence interval 1.87-19.77).ConclusionsOur data indicate actively ongoing B cell-mediated immunity against brain antigens in a subset of MS patients that may be causative of clinical relapses and provide new diagnostic and therapeutic options for a subset of patients.

Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis.

In multiple sclerosis (MS) lymphoid follicle-like aggregates have been reported in the meninges of patients. Here we investigated the functional relevance of B cell infiltration into the central nervous system (CNS) in MP4-induced experimental autoimmune encephalomyelitis (EAE), a B cell-dependent mouse model of MS. In chronic EAE, B cell aggregates were characterized by the presence of CXCL13(+) and germinal center CD10(+) B cells. Germline transcripts were expressed in the CNS and particularly related to TH17-associated isotypes. We also observed B cells with restricted VH gene usage that differed from clones found in the spleen. Finally, we detected CNS-restricted spreading of the antigen-specific B cell response towards a myelin and a neuronal autoantigen. These data imply the development of autonomous B cell-mediated autoimmunity in the CNS in EAE - a concept that might also apply to MS itself.

KIR4.1 Antibodies as Biomarkers in Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune disease caused by the infiltration of autoreactive lymphocytes into the central nervous system (CNS). The influence of B cells has been underestimated for a long time. Recently, it has become more apparent that B cells can fundamentally contribute to the pathogenesis of MS in terms of antigen presentation, co-stimulation, cytokine production, ectopic lymphoneogenesis, and antibody secretion (1–3). Along these lines, B cell depletion in MS patients with a relapsing-remitting disease course was able to reduce inflammatory brain lesions and clinical relapses (4) and B cell activation influences T cell polarization in the animal model of MS (5). Great effort has been made to utilize B cells for dividing patients into subgroups and to predict treatment responses in individual patients to specific drugs (6). Especially the detection of autoantibodies against CNS antigens is a crucial research subject due to its capability to be used as disease-specific biomarkers for the diagnosis and prognosis of MS. To this end, in a clinical study published in 2012 in the New England Journal of Medicine, Srivastava and colleagues screened serum samples aiming to identify CNS-specific antibodies in MS (7). The authors identified the glial potassium channel KIR4.1 as one of the serum antibody targets in MS patients. Subsequently, an enzyme-linked immunosorbent assay (ELISA) large-scale screening with 397 MS patients, 329 persons with other neurological diseases, and 59 healthy donors was performed. Antibodies against KIR4.1 were observed in 46.9% of patients with MS, but were essentially absent in people with other neurological diseases and healthy donors. Based on these data, Srivastava et al. concluded that KIR4.1 is a CNS-specific target of the autoantibody response in a subgroup of patients with MS. We would like to carefully raise the possibility that the serum IgG reactivity against KIR4.1 might present an epiphenomenon and there could be an additional target for autoreactive B cells beyond the CNS. It has to be considered that KIR4.1 is not only expressed in the CNS, but also on parietal cells of the gastric mucosal epithelium (8). The KIR4.1 channel expression is essential for the parietal cell control of acid secretion (9, 10). It is well known that there is a connection between the gastrointestinal immune response and CNS autoimmunity (11, 12). There are several possible mechanisms including molecular mimicry, determinant spreading, or bystander activation explaining the link between gastrointestinal immune response and MS (13). As recently published by Banati et al. (11), antibodies against parietal cells are also present in the sera of patients with MS. The presence of anti-parietal cell antibodies was most frequently associated with gastrointestinal complaints of the MS patients (11). Thus, it is possible that an impaired gastric mucosa, which can appear in cases of a frequent corticosteroid therapy, could extensively expose the parietal cell KIR4.1 to B cells. Corticosteroids augment gastroduodenal permeability and high doses are associated with macroscopic mucosal lesions (14). Therefore, we consider it as necessary to include the history of gastrointestinal complaints in the anamnesis of the study subjects before defining KIR4.1 as a CNS target of serum antibodies. In the setting of patients with an initial demyelinating event who have not received corticosteroids, one could readily exclude an impact of the MS medication on the gut. Finding anti-KIR4.1 antibodies in such patients in combination with gut impairments could raise the possibility that the gut itself is also a target of the autoimmune process. While further investigations are awaited to clearly analyze the impact of immune responses against antigens of gastric parietal cells on the finding of KIR4.1-reactive antibodies in patients with MS, the diagnostic potential of serum KIR4.1 antibodies may be highly clinically relevant. Therefore, the sensitivity and specificity of the initial findings of serum KIR4.1 antibodies in a subgroup of MS patients should be confirmed in studies from independent researchers. Furthermore, the pathogenicity of KIR4.1 antibodies needs to be clarified. Only 2 of 19 serum IgG positive MS patients also had detectable levels of KIR4.1 antibodies in the cerebrospinal fluid (CSF) (7). By analogy, aquaporin-4 antibodies were not readily detected in the CSF in neuromyelitis optica patients (15). Immunhistochemical investigations by Lennon et al. (16) showed that serum aquaporin-4 antibodies in patients with neuromyelitis optica bind to CNS tissue as well as to parietal cells of the gastric mucosa (16). However, it is beyond doubt that aquaporin-4 antibodies are highly pathogenic in neuromyelitis optica since they were studied extensively. Kinoshita and colleagues have demonstrated the pathogenic role of aquaporin-4 antibodies in vivo (17). They obtained serum IgG from patients with neuromyelitis optica and detected astrocytic damage in rats after passive transfer of aquaporin-4 containing IgG. In vivo experiments of this kind could delineate whether KIR4.1 antibodies are only a disease marker or if they play a central pathogenic role. To this end, KIR4.1 antibody containing serum could be injected into mice and subsequent histological and immunohistochemical investigations could show potential resulting pathological effects in CNS and gut. Furthermore, astrocytes should be incubated with KIR4.1 antibody containing serum and cell viability assays may demonstrate whether KIR4.1-reactive IgG induces astrocytic cytotoxicity.

The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis

There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.

The role of CEA-related cell adhesion molecule-1 (CEACAM1) in vascular homeostasis.

Carcinoembryonic antigen (CEA)-related cell adhesion molecules belong to the immunoglobulin superfamily, are expressed in a broad spectrum of tissues and cell types and exert context-dependent activating as well as inhibitory effects. Among these molecules, the CEA-related cell adhesion molecule-1 (CEACAM1) is a transmembrane molecule with an extracellular, a transmembrane and a cytoplasmic domain. The latter contains immunoreceptor tyrosine-based inhibitory motifs and functions as a signaling molecule. CEACAM1 can form homo- and heterodimers which is relevant for its signaling activities. CEACAM1 acts as co-receptor that modulates the activity of different receptor types including VEGFR-2, and B and T cell receptors. CEACAM1 is expressed in endothelial cells, in pericytes of developing and newly formed immature blood vessels and in angiogenically activated adult vessels, e.g., tumor blood vessels. However, it is either undetectable or only weakly expressed in quiescent blood vessels. Recent studies indicated that CEACAM1 is involved in the regulation of the endothelial barrier function. In CEACAM1−/− mice, increased vascular permeability and development of small atherosclerotic lesions was observed in the aortae. CEACAM1 is also detectable in activated lymphatic endothelial cells and plays a role in tumor lymphangiogenesis. This review summarizes the vascular effects of CEACAM1 and focuses on its role in vascular morphogenesis and endothelial barrier regulation.

Visualization of endothelial barrier damage prior to formation of atherosclerotic plaques

En-face fat staining is frequently used to visualize atherosclerotic lesions. This method, however, is not suitable to visualize endothelial barrier damage prior to microscopically detectable morphological alterations of the arterial wall such as sub-endothelial lipid deposition. To enable the investigation of early endothelial barrier damage and in particular the initial steps of atherosclerosis, a new method has to fulfill three requirements: (i) easy and fast to perform, (ii) low cost of applicability without requirement for highly sophisticated technical equipment, and (iii) reliable reproducibility of valid results. To this end, we used intracardial Evans blue dye injection after washout of blood and measured dye deposition within the aortic wall as a parameter of endothelial barrier leakiness, which is recognized as one of the earliest signs of atherosclerotic plaque formation. These analyses were performed in ApoE−/−, LDL receptor−/− and Cc1−/− mouse models which have been reported to develop aortic plaques with or without high cholesterol diet. Our data show that sub-endothelial dye deposition is a reliable and reproducible readout parameter to assess endothelial barrier damage. Along these lines, measurements of aortic intima areas with Evans blue deposition in relation to total intima circumference enabled quantitative assessments of the results. Our technique enables the imaging of endothelial barrier damage prior to detectable aortic lipid deposition and plaque development. Thus, it will facilitate the detection of the initial vascular pathogenetic processes that lead to cardiovascular diseases. It will also enable the testing of new drugs and therapeutic procedures to prevent these disorders.