Wolfgang E. F. Klinkert

Neuronal or glial progeny: Regional differences in radial glia fate

The precursor function of the ubiquitous glial cell type in the developing central nervous system (CNS), the radial glia, is largely unknown. Using Cre/loxP in vivo fate mapping studies, we found that radial glia generate virtually all cortical projection neurons but not the interneurons originating in the ventral telencephalon. In contrast to the cerebral cortex, few neurons in the basal ganglia originate from radial glia, and in vitro lineage analysis revealed intrinsic differences in the potential of radial glia from the dorsal and ventral telencephalon. This shows that the progeny of radial glia not only differs profoundly between brain regions but also includes the majority of neurons in some parts of the CNS.

Real-time imaging reveals the single steps of brain metastasis formation

Brain metastasis frequently occurs in individuals with cancer and is often fatal. We used multiphoton laser scanning microscopy to image the single steps of metastasis formation in real time. Thus, it was possible to track the fate of individual metastasizing cancer cells in vivo in relation to blood vessels deep in the mouse brain over minutes to months. The essential steps in this model were arrest at vascular branch points, early extravasation, persistent close contacts to microvessels and perivascular growth by vessel cooption (melanoma) or early angiogenesis (lung cancer). Inefficient steps differed between the tumor types. Long-term dormancy was only observed for single perivascular cancer cells, some of which moved continuously. Vascular endothelial growth factor-A (VEGF-A) inhibition induced long-term dormancy of lung cancer micrometastases by preventing angiogenic growth to macrometastases. The ability to image the establishment of brain metastases in vivo provides new insights into their evolution and response to therapies.

Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions

The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.

T cells become licensed in the lung to enter the central nervous system

The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.

Anti‐inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis: inhibition of monocyte transendothelial migration

In order to analyze a putative immunomodulatory effect of NGF in experimental autoimmune encephalomyelitis (EAE) of the Lewis rat, we transduced myelin basic protein (MBP)‐specific CD4+ T cells with a recombinant retrovirus encoding NGF. These TMBPNGF cells secreted high levels of NGF, along with an unaltered Th1‐like cytokine pattern. Transfer studies showed that TMBPNGF cells were unable to mediate clinical EAE, when transferred alone, and, more important, they efficiently suppressed induction of clinical EAE by non‐transduced MBP‐specific T cells (TMBP cells). In contrast, NGF transduced ovalbumin‐specific T cells, which secreted high NGF levels, did not affect EAE induction. Suppression of clinical EAE by TMBPNGF cells was associated with a general reduction of inflammatory CNS infiltrates, with a most pronounced decrease of the monocyte/macrophage component. Using a culture model of the endothelial blood‐brain barrier (BBB), we found that NGF directly acts on blood‐derived monocytes via the p75 NGF receptor, thus interfering with monocyte migration through the activated BBB endothelium. Our data establish NGF as an anti‐inflammatory mediator interfering with T cell mediated autoimmune disease in the CNS. They further point to monocyte migration through blood vascular endothelium as one possible mechanism of NGF action.

The activation status of neuroantigen-specific T cells in the target organ determines the clinical outcome of autoimmune encephalomyelitis

The clinical picture of experimental autoimmune encephalomyelitis (EAE) is critically dependent on the nature of the target autoantigen and the genetic background of the experimental animals. Potentially lethal EAE is mediated by myelin basic protein (MBP)–specific T cells in Lewis rats, whereas transfer of S100β- or myelin oligodendrocyte glycoprotein (MOG)–specific T cells causes intense inflammatory response in the central nervous system (CNS) with minimal disease. However, in Dark Agouti rats, the pathogenicity of MOG-specific T cells resembles the one of MBP-specific T cells in the Lewis rat. Using retrovirally transduced green fluorescent T cells, we now report that differential disease activity reflects different levels of autoreactive effector T cell activation in their target tissue. Irrespective of their pathogenicity, the migratory activity, gene expression patterns, and immigration of green fluorescent protein+ T cells into the CNS were similar. However, exclusively highly pathogenic T cells were significantly reactivated within the CNS. Without local effector T cell activation, production of monocyte chemoattractants was insufficient to initiate and propagate a full inflammatory response. Low-level reactivation of weakly pathogenic T cells was not due to anergy because these cells could be activated by specific antigen in situ as well as after isolation ex vivo.

TNF-α receptor fusion protein prevents experimental auto-immune encephalomyelitis and demyelination in Lewis rats: an overview

To explore the therapeutic use of TNF-alpha inhibitors in human inflammatory demyelinating diseases we examined the effect of a recombinant TNFRp55 protein constructed by fusing TNFRp55 extracellular domain cDNA to a human IgG1 heavy gene fragment containing the hinge and constant domains CH2 and CH3 (TNFRp55-IgG1) in diverse experimental model systems representing inflammation and inflammatory demyelination of encephalitogenic T cells in vivo. In EAE actively induced by immunization of Lewis rats with MBP, a single dose of TNFRp55-IgG1 protected the recipient animals from clinical signs. Interestingly, the treatment neither prevented the formation CNS infiltrations, nor did it alter the cellular composition of the infiltrates. In EAE transferred by MBP specific activated T line cells, a model of inflammatory (not demyelinating) brain disease, the inhibitors therapeutic effect on clinical disease was also striking achieving almost complete protection even after repeated transfers of encephalitogenic T cells. Finally, the recombinant inhibitor was also protective in Lewis rats with demyelinating experimental autoimmune panencephalitis produced by combined transfer of panencephalitogenic T cells and demyelinating monoclonal antibody specific for MOG. In this system, the T cells are of low encephalitogenic activity, but open the blood-brain barrier for the demyelinating immunoglobulins. The fusion protein treatment, however, prevented the formation of inflammatory lesions and demyelination. The strong therapeutic effect of the recombinant chimeric TNF-alpha inhibitor in three models of myelin specific autoimmunity raises hopes as to TNF-alpha directed therapy of human diseases like MS.

Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.

Secretion of brain-derived neurotrophic factor by glatiramer acetate-reactive T-helper cell lines: Implications for multiple sclerosis therapy

Treatment with glatiramer acetate (GA) is thought to induce an in vivo change of the cytokine secretion pattern and the effector function of GA-reactive T helper (TH) cells (TH1-TH2-shift). Current theories propose that GA-reactive TH2 cells can penetrate the CNS, since they are activated by daily immunization. Inside the CNS, GA-reactive T cells may cross-react with products of the local myelin turnover presented by local antigen-presenting cells (APCs). Thus, some of the GA-specific TH2 cells may be stimulated to release anti-inflammatory cytokines inhibiting neighbouring inflammatory cells by a mechanism called bystander suppression. We demonstrate that both GA-specific TH2 and TH1 cells produce the neurotrophin brain-derived neurotrophic factor (BDNF). To demonstrate that GA-reactive T cells produce BDNF, we analyzed GA-specific, long-term T-cell lines (TCLs) and used a combination of reverse-transcription PCR and two specially designed techniques for BDNF protein detection: one was based on ELISA of supernatants from co-cultures of GA-specific TCLs plus GA-pulsed antigen-presenting cells, and the other, on the direct intracellular staining of BDNF in individual T cells and flow-cytometric analysis. The different assays and different TCLs yielded similar, consistent results. All GA-specific TH1, TH2 and TH0 lines could be stimulated to produce BDNF.

Blood-borne soluble protein antigen intensifies T cell activation in autoimmune CNS lesions and exacerbates clinical disease

We explored the effect of i.v. soluble antigen on autoaggressive, myelin basic protein-specific effector T cells within their target organ during acute experimental autoimmune encephalomyelitis (EAE). Intravital two-photon imaging revealed that i.v. autoantigen reached the CNS and was taken up and processed by antigen-presenting cells within 30 min after injection. The exogenous autoantigen dramatically changed the motility and function of autoreactive effector T cells within the EAE lesions: T cells that had been cruising through the tissue slowed down and became tethered to local antigen-presenting cells within 1 h. One hour later, the effector T cells massively produced proinflammatory cytokines and up-regulated membranous activation markers. This strong activation of the T cells boosted CNS inflammation and aggravated clinical disease. Postactivated effector and resting memory T cells specific for a non-CNS antigen (ovalbumin) were recruited to EAE lesions and moved there without contacting antigen-presenting cells. These cells were similarly arrested and activated after i.v. infusion of ovalbumin, and they also exacerbated clinical disease. Our data are relevant for autoantigen-based therapies of autoimmune disorders. Further, the study indicates how brain unrelated antigens (microbial components) leaking into the chronically inflamed CNS through the bloodstream might trigger relapses in multiple sclerosis.