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Presence of B7–2 (CD86) and lack of B7–1 (CD(80) on myelin phagocytosing MHC-II-positive rat microglia is associated with nondestructive immunity in vivo¹

INGO BECHMANN^*2, SUSANNE PETER^*, MARTIN BEYER, ULRIKE GIMSA^* and ROBERT NITSCH^*

^* Institute of Anatomy, Department of Cell and Neurobiology and
Clinic of Neurology, Department of Clinical Neuroimmunology, Humboldt-University Hospital Charité, 10098 Berlin, Germany

²Correspondence: Institute of Anatomy; Department Cell and Neurobiology, Humboldt University Hospital Charité; 10098 Berlin, Germany. E-mail: ingo.bechmann{at}charite.de

SPECIFIC AIMS

Myelin-specific autoimmune T cells can induce both destructive and neuroprotective autoimmunity and it is likely that differences in peripheral T cell stimulation account for the induction of the one vs. the other response. To unravel such differences, we studied the expression of MHC class II and costimulatory B7–1/B7–2 molecules on myelin phagocytosing microglia after rat entorhinal cortex lesion, a model of axonal degeneration followed by reactive synaptogenesis without destructive autoimmunity.

PRINCIPAL FINDINGS

1. MHC class II expression on myelin phagocytosing microglia
A previously established technique of phagocytosis-dependent labeling was applied to search for MHC-II-positive myelin phagocytosing microglia. The dextran- and biotin-conjugated tracer Mini Ruby was used to label perforant path axons. This myelinated fiber tract connects the entorhinal cortex with the hippocampus. Subsequent stereotactic entorhinal lesion induces degeneration of these fibers in their termination zones, i.e., the middle molecular layer of the dentate gyrus (MML). Ipsi- and contralateral fibers to the entorhinal cortex are retrogradely affected by such lesions. Uptake of the fluorescent axon debris allowed identification of phagocytosing cells using (immune) double-fluorescence microscopy. The phagocytosed material was localized within MHC-II-positive cells exhibiting the typical morphology of activated microglia. Indeed, all of these MHC-II-positive cells were identified as microglia/macrophages using isolectin-B4 labeling. Never was MHC-II found on GFAP-positive astrocytes. The first MHC-II-positive microglial cells appeared around 6 days postlesion (dpl) in the MML. In this zone, where only anterograde degeneration occurs, the number of immune-positive cells as well as their immune reactivity clearly increased until about 12 dpl, but disappeared at day 40 postlesion. At this time, MHC-II-positive microglia were still found in the alveus, the fimbria, and in the anterior and posterior commissure, where entorhinal lesions induce anterograde and retrograde degeneration.

2. Lack of B7–1 on myelin phagocytosing microglia
Several anti-B7–1 antisera/antibodies did not detect glial cells in the brain parenchyma after entorhinal cortex lesion (ECL; Fig. 1a , b , c ). However, cell populations outside of the parenchyma that are known to express B7–1, such as endothelial and perivascular cells (Fig. 1b ) as well as leucocytes inside of blood vessels (Fig. 1c ), were readily detected. In some lesioned and control animals, randomly distributed single B7–1-positive microglia and macrophages were found (Fig. 1d ).

View larger version (104K): [in this window] [in a new window]   Figure 1. B7–1/B7–2 expression after ECL. No B7–1-positive glial cells are found in the denervated hippocampus of controls (a) and after lesion (b, c). The different width of the molecular layers in panels a, b is due to well-described shrinkage after entorhinal lesion. Some endothelial cells and perivascular macrophages exhibit B7–1 staining (arrows in panel b). Immune-positive leucocytes are seen in blood vessels (arrows in panel c) indicating binding of the primary antibody. In some lesioned and control animals, randomly distributed B7–1-positive cells were found in circumscribed areas. These cells exhibited the typical morphology of microglia and macrophages (arrows in panel d). Randomly distributed, highly ramified B7–2-positive microglia were found in the white matter of control and lesioned animals (arrows in panel e). After entorhinal lesion, B7–2 expression was enhanced in the hippocampal zone of anterograde degeneration, the middle molecular layer (MML) (f–h). This expression peaked about day 5 postlesion and remained visible for as long as several weeks after lesion. Original magnification: a, b) 200x; c); 400x; d, e) 600x; f–h) 400x.

3. Enhanced long-time B7–2 expression on microglia
B7–2 was found at low levels on many randomly distributed ramified cells in the white matter of lesioned and control animals (Fig. 1e ). As anticipated by the morphology of these cells, they were identified as microglia using isolectin-B4 labeling. After entorhinal lesion, a strong increase in B7–2 expression occurred in the termination zone of the perforant path (Fig. 1f , g , h ) and in zones of combined anterograde and retrograde degeneration (alveus, fimbria, anterior and posterior commissure). This expression reached its peak at about 5 dpl and was still enhanced after several weeks. Double-labeling of B7–2 and GFAP revealed no colocalization in lesioned and control animals.

4. Invasion of CD4/B7–2-positive /ß t cells without microglial activation
In zones of axonal degeneration, homing T cells were found at the ultrastructural level (Fig. 2a , b ). Immunocytochemistry using various antibodies revealed single CD4-positive T cells in the MML in the first days after lesion (Fig 2c ). CD8-positive T cells were not detected. In zones of combined anterograde and retrograde degeneration, R73 immune staining identified numerous /ß T cells. Infiltration of such T cells was still evident at 90 dpl (Fig. 2e ) and was accompanied by B7–2 expression on morphologically resting, ramified microglia (Fig. 2d ). The T cells were also found to express B7–2 (Fig. 2f , g ).

View larger version (134K): [in this window] [in a new window]   Figure 2. T cell infiltration after ECL. After entorhinal lesion, homing T cells were found in the zone of anterograde degeneration at the ultrastructural level (a, b). Only single CD4-positive cells were found in the zone of anterograde degeneration (arrow in panel c) at any time after lesion. B7–2 expression on infiltrating CD4-positive T cells. In the alveus, a zone of retrograde degeneration after ECL, a long lasting B7–2 expression was found on microglia (arrowheads in panel d) and on leukocyte-like cells (arrows in panel d). This B7–2 expression was accompanied by infiltration of /ß R73-positive T cells (e). Note the ramified morphology of the microglia indicating their resting state despite T cell infiltration. The small B7–2-positive cells, as indicated by arrows in panel d, were identified as CD4-positive T cells using double-fluorescence (f, g). The microglia-like B7–2-positive cell in panel f (arrowhead) cannot be seen in panel g, indicating the selectivity of the fluorescence filters. Original magnification: 600x (c, d).

CONCLUSIONS AND SIGNIFICANCE

Autoimmune demyelinating diseases such as multiple sclerosis (MS) and its animal model, experimental allergic encephalomyelitis (EAE), are induced by autoimmune T cells. By unknown stimuli, these T cells invade the central nervous system (CNS) where they activate microglia to secret proinflammatory molecules and to phagocytose myelin, which then is presented upon B7–1 costimulation. This, in turn, can contribute to further T cell recruitment, activation, and differentiation (see schematic diagram). Blockade of B7–1, therefore, inhibits disease induction and abrogates ongoing disease. It was the aim of this study to understand why mechanically induced axonal degeneration does not induce destructive autoimmunity despite myelin being phagocytosed and presented on local antigen-presenting cells. On the contrary, in this setting T cells directed against myelin epitopes contribute to axonal survival, rendering application of such T cell clones a therapeutic option after CNS trauma. This requires, however, a better understanding of the signals inducing destructive vs. the signals inducing protective autoimmunity to exclude development of autoimmune disease after such treatment.

As shown in this study, the different immune responses to myelin in EAE/MS vs. mechanical injury are, in contrast to previous explanations, not due to 1) lack of infiltrating T cells and 2) lack of MHC-II expression. The striking difference we found is that myelin obviously is not presented in a MHC-II/B7–1, but in a MHC-II/B7–2 context. Several lines of evidence indicate that whereas B7–1 is related to destructive autoimmunity, B7–2 expression induces protective, or at least less harmful, T cell responses. Our finding of myelin phagocytosing microglia exhibiting a MHC-II⁺/B7–2⁺/B7–1^- phenotype might thus be the reason why such axonal degeneration is not followed by harmful autoimmunity despite presentation of immunogenic myelin epitopes. It is noteworthy, that we found T cells present in the brain even long after the experimental lesion, but in contrast to autoimmune brain disease, they did not induce microglial activation. Conversely, microglia exhibited a ramified morphology while expressing both MHC-II and B7–2 in the presence of these CD4-positive T cells. It is currently unclear which function can be attributed to B7–2 expressed on such invading /ß T cells. Nevertheless, it indicates a reverse B7–2/CTLA4 signal of T cells finally inducing a resting state in microglia.

The data presented here show that in contrast to the destructive immune response in MS and EAE, axonal degeneration lacks a signal to induce microglial B7–1 expression and attracts CD4-positive /ß T cells. These cells were rare in the zones of anterograde degeneration, where axons are prone to die, but were numerous with a long-lasting presence in zones of retrograde degeneration where fibers are still connected to their pericaryon, allowing survival and regrowth under certain conditions. These findings indicate a protective role of T cells addressed as benign autoimmunity. Our approach allows future studies to analyze how T cells can contribute to axonal protection after central nervous system injury. This might include secretion of neurotrophins and changes in electrophysiological properties such as electrical insolation.

View larger version (30K): [in this window] [in a new window]   Figure 3. Interaction between T cells and local antigen-presenting cells: protective vs. destructive immunity. Left site: axonal degeneration induces MHC-II and B7–2 expression on microglia (MG). Infiltrating T cells interact with antigen-presenting microglia and might receive a CTLA-4 mediated signal either by microglia or by other T cells, resulting in ‘peaceful coexistence’ of both cell types in the tissue. T cells might then contribute to axonal protection by secretion of neurotrophins and electrical insolation of axons. Right site: upon unknown stimuli outside of the brain, myelin-specific T cells proliferate, differentiate, and invade the CNS. T cell-mediated signals induce B7–1 expression on microglia, which further stimulates the T cells via CD28. Secretion of cytotoxins and bystander lysis by either cell type leads to destructive immunity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0563fje ; to cite this article, use FASEB J. (February 26, 2001) 10.1096/fj.00-0563fje

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