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Morphological aspects of spinal cord autoimmune neuroprotection: colocalization of T cells with B7–2 (CD86) and prevention of cyst formation¹

Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel

²Correspondence: Department of Neurobiology, Arison Bldg. 221, The Weizmann Institute of Science, 76100 Rehovot, Israel. E-mail: michal.schwartz{at}weizmann.ac.il

SPECIFIC AIMS

Spinal cord injury often leads to massive degeneration which is greatly in excess of that caused directly by the insult. Systemic passive transfer of autoimmune T cells was shown by our group to reduce the extent of degeneration, as confirmed functionally, anatomically, and by image analysis. The present study was undertaken to characterize the interrelationships between T cells, macrophages/microglia, the expression of costimulatory molecules such B7–2 (also known as CD86), and resident cells within the damaged spinal cord after contusion with or without treatment by systemic injection of T cells directed against myelin basic protein (MBP). The effect of the T cells on other injury-related events such as cyst formation was also studied.

Spinal cord injury, like any central nervous system (CNS) insult, frequently has a devastating outcome because of the failure of CNS axons to regenerate. The final neuronal loss after such an injury, however, is often substantially greater than would be expected from the severity of the insult. This ‘secondary degeneration’ has been the target of wide-ranging research efforts in an attempt to enhance the recovery from the injury. Therapeutic approaches that aim to diminish secondary degeneration are collectively termed neuroprotection. A pharmacological neuroprotective approach is likely to have side effects, due to inevitable interference with the normal physiological functioning of these compounds.

An alternative neuroprotective strategy without this inherent disadvantage is based on increasing the ability of the neurons to cope with injurious conditions. One such strategy is autoimmune protection, an immunological approach, based on an unexpected recent discovery by our group. It was found that ‘autoimmune T cells’, i.e., T cells directed against myelin-associated CNS self-antigens, whether evoked spontaneously by the injury or boosted by passive or active immunization, are capable of targeting themselves to the lesion site and reducing the injury-induced spread of damage.

PRINCIPAL FINDINGS

We show here that passive transfer of anti-MBP T cells (T_MBP) leads to protection of neurons that escaped the primary insult. Within the damaged spinal cord, these T cells were found to colocalize with structures that look like precursors of cysts. Accumulation of T cells in such areas is associated with the up-regulation of B7–2 costimulatory molecules, immunological surface molecules known to play a role in the dialog between T cells and their effector cells.

1. Autoimmune T cells promote tissue preservation and prevention of cyst formation
Inbred adult female Lewis rats were subjected to spinal cord contusion and were examined morphologically 7, 14, or 28 days or 21 wk after the injury. Rats in the experimental group were injected with 10⁷ T_MBP. Functional recovery was evaluated by assessing motor activity in an open field. Histological, anatomical, and immunochemical analysis of contused spinal cords at various times after injury and treatment showed better tissue preservation in the T_MBP-treated cords than in the spinal cords of control rats injected with phosphate-buffered saline (PBS). In addition to the significant preservation of axons, the most striking observation was the minimal cyst development in the T_MBP-treated spinal cord as opposed to the massive cysts that developed in the controls (Fig. 1 ).

View larger version (105K): [in this window] [in a new window]   Figure 1. Colocalization of B7–2-expressing cells and T cells on the 28th day after injury in TMBP-treated and PBS-treated rats. Micrographs are from TMBP-treated rats (A, B) and PBS-treated controls (C, D). A1) B7–2-expressing cells are present in large numbers, almost filling the precyst-like structure. B1) Boxed area from panel A1. B7–2-labeled cells only, without bright field. A2) The same field as in panel A1 from an adjacent slice, stained for TCR. B2) Boxed area at high magnification. C) Necrotic abrasion, shown by arrow. This section was taken from a PBS-treated spinal cord at a distance of 6 mm from the site of injury. No staining for B7–2 was detected in this structure. D) A well-formed encapsulated cyst containing no cells. This section was taken at a distance of 8 mm from the site of injury in a control rat.

2. B7–2 expression by autoimmune T cells
The T_MBP were found to be localized in areas of the spinal cord containing structures that seem to have the potential to develop into cysts in the absence of therapeutic intervention; these areas were also found to be strongly positive for B7–2 costimulatory molecules (Fig. 1) . In contrast, hardly any B7–2 immunoreactivity could be detected in the spinal cords of PBS-treated controls. Close examination of individual cells after immunocytochemical double staining revealed that T cells, presumably including both those that are endogenous and those that were transferred, were among the cells labeled with B7–2. Microglia/macrophages were also positive for B7–2.

3. T cells can up-regulate in vitro expression of B7–2
Our in vivo studies showed that the injection of T_MBP caused up-regulation of B7–2 on T cells and macrophages/microglia at the lesion site. To determine whether T_MBP can directly activate microglia to express B7–2, microglia-enriched populations were cocultured either alone or together with activated T_MBP. After 24 h of culturing, the cells were double-labeled with anti-ED-1 and anti-B7–2 monoclonal antibodies and analyzed by confocal microscopy. Abundant expression of B7–2 cells was seen in microglia cocultured with T_MBP; larger numbers of T cells caused greater proliferation of microglia, as indicated by ED1-positive cells (Fig. 2 ).

View larger version (115K): [in this window] [in a new window]   Figure 2. In vitro coculturing of primary microglia with activated TMBP cells results in induced expression of B7–2. Confocal microscopy shows double staining of microglia stained for ED-1 and B7–2: a, b) control microglia, without added TMBP; c, d) microglia 24 h after coculturing with TMBP (2x103/well); e, f) microglia 24 h after coculturing with TMBP cells (2x104/well). For more details, see Materials and Methods. The data represent the results of three independent experiments, each performed in triplicate.

CONCLUSIONS AND SIGNIFICANCE

Previous studies from our laboratory have shown that systemic injection of autoimmune T cells, immediately after severe contusion (T8-T9) of the rat spinal cord, promotes recovery of locomotor activity, a fivefold increase in the number of intact axons descending from the red nucleus, and a significant increase in the mass of spared spinal cord. The main conclusion was that the effect of these autoimmune T cells on recovery results from the protection of neurons whose axons had escaped the primary lesion.

In humans, spinal cyst formation (or syringomyelia) is a condition characterized by the presence in the spinal cord of fluid-filled longitudinal cavities lined by dense gliogenous tissue. These cysts interrupt neural pathways in the central part of the spinal cord and are often associated with progressive myelopathy. Previous work from our laboratory has shown that T_MBP can prevent glial scarring and the development of syringomyelia. We further show here that the presence of T_MBP appears to be colocalized with up-regulated expression of B7–2 costimulatory molecules within the site of the lesion and caudally to it. The heightened expression of B7–2 is associated, at least in part, with the accumulation of T cells (either endogenous or adaptively transferred). These findings suggest that the induction of B7–2 expression plays an important role in preserving neuronal tissue and may be associated with the neuroprotective phenotype of T_MBP (see scheme, Fig. 3 ).

View larger version (57K): [in this window] [in a new window]   Figure 3. Proposed sequence of events in which B7–2 up-regulation presumably leads to neuroprotection. CNS trauma elicits an endogenous systemic T cell-mediated autoimmune response. The T cells infiltrate the CNS and participate in the dialog with resident microglia. These T cells, which probably include both Th1 and Th2, undergo paracrine and autocrine interaction with the microglia, resulting in some benefit to the damaged tissue. This benefit is, however, apparently insufficient to counteract the self-destructive effects of the hostile environment produced by the increase in nitric oxide and excitatory amino acids (EAA), as well as by other compounds, collectively resulting in the spread of damage (A). Boosting of the autoimmunity by passive transfer of autoimmune T cells directed against MBP results in a greater accumulation of T cells at the lesion site. Through their local interaction with microglia, these T cells cause up-regulation of B7–2 in both the microglia and the T cells, resulting in T cells with a neuroprotective phenotype (Th-NP). These T cells are able to counteract the destructive effects, and the overall result is tissue rescue (B).

The dramatic increase in B7–2 expression was associated with an immune response that was neuroprotective in its effect. These observations prompt us to suggest that resident macrophages (microglia) might be activated, by their interaction with activated T cells, to express B7–2. Signals derived from preactivated CD4+ T cells are thought to be essential for such microglial activation. In vitro, T_MBP-activated cells were found to induce the expression of costimulatory molecules on cultured rat microglia, possibly via interferon activity. The up-regulation of B7–2 might lead to an enhanced ability of microglia to support antigendependent T cell activation. Recently it was shown that antigen-specific interactions between microglia and Th1 cells ex vivo induce the expression of MHC class II and adhesion/costimulatory molecules, resulting in activation of Th1 or naive T cells but not of Th2. Those observations are in line with our in vivo and in vitro findings that interactions between activated T_MBP and resting microglia can stimulate the expression of B7–2 costimulatory molecules on microglia. These findings suggest that infiltration of the CNS by preactivated T cells may cause resident microglia to mature into competent antigen-presenting cells that can sustain CD4+ T cell responses within the CNS. It is likely that T cell-activated microglia are capable of contributing to the local Th1/Th2 balance. These mutual interactions might influence the outcome of CNS inflammation in a way that is either beneficial or destructive to the damaged tissue.

In most tissues, injury-induced damage triggers a cellular immune response that acts to repair the tissue and preserve its homeostasis. This response has been attributed to microglia and macrophages, which comprise the innate arm of the immune system. There are several hypotheses concerning the effects of macrophages on the site of injury. In their role as professional scavengers, the macrophages have two main functions: the first has to do with the cytolysis and phagocytosis of dead and dying cells and their debris in damaged nervous tissue, and the second concerns their stimulatory effect on the reconstructive phase of healing. It therefore seems that macrophages may exhibit either a beneficial or a harmful effect, depending on their number, state of activation, cellular context, and whether the tissue requires rescue or regrowth. It is thus reasonable to assume that in the presence of T_MBP, the dialog of the damaged CNS with resident microglia/macrophages will potentially be of benefit to the neuronal tissue (Fig. 3) .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0550fje ; to cite this article, use FASEB J. (February 26, 2001) 10.1096/fj.00-0550fje

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