We next used confocal laser microscopy with immunofluorescence detection of the astrocytic marker GFAP and the neuronal marker MAP2 to characterize the effects of tat-transfected RAW264.7 cells on the cytoarchitecture of the hippocampal slice culture (Fig. 3). Using a double labelling technique, it was also possible to identify cells that had accumulated propidium iodide during the experiment. Untreated cultures revealed a clear meshwork of GFAP-positive astrocytic processes surrounding neurones, with no evidence of propidium iodide uptake (Fig. 3a). Twenty-four h after co- incubation with Tat-producing RAW264.7 cells, the astrocytic matrix was completely destroyed (Fig. 3e) and propidium iodide-positive nuclear staining was evident throughout the culture. At very high power, nuclear propidium iodide staining revealed clear signs of chromatin clumping and the formation of small inclusions similar to those described during apoptosis (Fig. 4). MAP2 staining of neuronal processes revealed clear healthy-looking neurones with well organized neuronal processes in untreated organotypic cultures (Fig. 3b). No propidium iodide staining was evident in these control cultures, however 24 h after incubation with Tat-producing RAW264.7 cells, intense propidium iodide staining was present in the neuronal cell fields (Fig. 3f). At this time point, MAP2 staining had almost completely disappeared with only a few neuronal processes still visible (Fig. 3f).
To characterize further the nature of the toxic effect slice cultures were incubated with conditioned medium from cultures of Tat-producing RAW264.7 cells. Although conditioned medium did not consistently result in propidium iodide labelling in all organotypic slice cultures, the cytoarchitecture of both GFAP- and MAP2- expressing cells was consistently found to be distorted significantly (Fig. 3c, d). Astrocytic processes appeared retracted and no longer tightly surrounded cells within principal neuronal cell layers (Fig. 3c), leaving large exclusion zones in the culture where no GFAP staining was evident. Similarly evidence of cytoplasmic and nuclear swelling in MAP2-expressing neurones indicated that these were also affected by the conditioned medium (Fig. 3d). These results suggest that conditioned medium from Tat producing RAW264.7 cells induced morphological changes in organotypic cultures but was not sufficient to reliably produce cell death. The pattern of damage seen with Tat-induced damage was not replicated by known glutamate receptor agonists (Fig. 5). Incubation of slice cultures with 10mM Glutamate, 10μM AMPA, 3μM kainate or 10μM NMDA produced typical regionally selective patterns of cell death. There was no evidence of glial cell death with any of the glutamate agonists tested, suggesting that toxicity of the Tat-induced factor is unlikely to be mediated via these receptors.
The results obtained by using a new model based on the co-culture of macrophages with organotypic hippocampal slice cultures, provides the first clear evidence that a soluble factor(s) may be responsible for the neurotoxicity produced by HIV-1 Tat protein in the brain, rather than Tat itself.
The reduced toxic effect of conditioned medium in our study may indicate that the Tat-induced factor is quite labile and hence is partially degraded when incubated with the slice cultures. Alternatively, the simultaneous presence of the slice cultures and Tat-producing macrophages may be required to express the full extent of this toxic effect, possibly involving some feedback between the two culture systems. This latter possibility could be related to the suggestion that amplificatory cellular responses involving astrocytes and neurones in addition to infected mononuclear cells may be responsible for the widespread neurodegeneration observed in ADC patients .
Recent studies have demonstrated the ability of Tat to induce activation of human monocytes and to stimulate their expression of a range of soluble factors including matrix metalloproteinase-9, transforming growth factor-β1, tumour necrosis factor (TNF)-agr;, interleukin (IL)-1β, IL-6 and IL-8 [33-35]. In addition, Lafrenie et al. showed that Tat treated monocytes displayed enhanced monocyte-endothelial cell adhesion and promoted profound disruption of endothelial cell monolayers, which could be mechanistically related to the profound cytoarchitectural disruption that we observed in the organotypic slice cultures [34,35]. As expression of TNF-agr;, IL-1 and IL-6 is induced in Tat-stimulated monocytes  and as all three cytokines have been implicated in Tat-induced neurotoxicity [12,36,37] one or more may be responsible for the neurotoxic damage observed in the current study. Recent work in our laboratory has revealed that TNF-agr; is not toxic when added on its own in organotypic slice cultures but that ir modulates toxicity induced by other excitotoxins significantly . Moreover, both PMA and LPS induce expression of TNF-agr; by RAW264.7 cells [39,40] yet neither PMA- nor LPS-treated RAW264.7 cells had neurotoxic effects when co-cultured with hippocampal slices. Increased production of TNF-agr; could act indirectly to potentiate damage; however, the primary damage must be triggered by other factors induced by Tat. It is also unlikely that either IL1-β or IL-6 are directly responsible for the primary damage as expression of both cytokines by RAW264.7 cells is induced co-ordinately with TNF-agr; in response to LPS [39-41]. It has been suggested that macrophages stimulated with cytokines or infected by HIV-1 may release other toxic substances such as quinolinic acid [42-44] thus killing neurones indirectly. Quinolinic acid is known to be selectively neurotoxic and does not cause the death of astrocytes because these contain enzymes which convert quinolinic acid to non-toxic metabolites [7,45]. A selectively neurotoxic agent such as quinolinate would result in a different pattern of propidium iodide staining than the one observed in our experiments and should not result in any astrocytic damage.
Our observations suggest that the toxicity observed with Tat-transfected cells or macrophages incubated with Tat may be primarily astrocytic. Propidium iodide images from early incubation times show widespread degeneration throughout regions that are not particularly rich in neurones but that contain mainly astrocytes (e.g., 7 h, Fig. 1c). Only at later time points (24 h) do the CA1 and CA3 regions, which are rich in pyramidal neurons, become affected (Fig. 1e). It therefore seems possible that the neurotoxicity produced by Tat could be secondary to a loss of astrocytic function, as neurones would be unlikely to survive long in the absence of glial support mechanisms. High resolution confocal microscopy of the propidium iodide signal revealed clear signs of nuclear fragmentation reminiscent of that described during apoptosis (see Fig. 4). This pattern of nuclear propidium iodide staining is never seen in organotypic cultures with classical necrotic insults such as incubation with glutamate receptor agonists (see Fig. 5) or ischaemic insults and suggests, at the very least, that the mode of death in this case is distinct from classical necrosis.
In summary, our work suggests that the HIV-1 regulatory protein Tat can induce the production of soluble toxic factor(s) from macrophages which is sufficient, if studied in a complex in-vitro system, to cause almost complete destruction of brain tissue. HIV-1 Tat has been found extracellularly and could therefore stimulate production of the neurotoxic factor by uninfected macrophages as well as by infected cells [12,15]. It is clear that if such mechanisms are operational in the brains of individuals with AIDS this could result in the widespread neurodegeneration characteristic of patients suffering ADC. We propose that the co-culture model described in this study can be used to identify HIV-1 induced macrophage-derived neurotoxic factors and to study the mechanisms by which these factors induce cell death in the brain.
The authors thank R. Alston and S. Bottoms for their help with confocal imaging.
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