Retroviral gene transfer, rapid selection, and maintenance of the immature phenotype in mouse dendritic cells

We used the retroviral vector PINCO [which expresses the green fluorescent protein (GFP) as a selectable marker], to infect growth factor-dependent immature D1 dendritic cells (DC). The efficiency of infection in different experiments was between 5 and 30%, but subsequent cell sorting led to a virtually homogeneous population of GFP-positive cells. Retroviral infection did not modify the immature DC phenotype, as shown by the low expression of major histocompatibility complex and co-stimulatory molecules. Furthermore, the GFP-positive D1 cells underwent full maturation after lipopolysaccharide treatment, as indicated by a high expression of cell-surface MHC and co-stimulatory molecules, and also by strong stimulatory activity in allogeneic mixed lymphocyte reaction. The high efficiency of this retroviral system, the rapidity of the technique, and the possibility to overcome in vitro selection make this method very attractive for the stable introduction of heterologous genes into proliferating immature mouse D1 cells. Furthermore, this approach is suitable for functional studies of new DC-specific genes involved in DC maturation and survival

The potential role of dendritic cells in immune-mediated inflammatory diseases in the central nervous system

Dendritic cells of the rat were studied immunohistochemically with MRC OX62 monoclonal antibody and using electron microscopy. In normal CNS, a small number of OX62+ cells was detected in the choroid plexus and meninges. These cells were absent from other CNS and peripheral nervous system sites studied. Dendritic cells were also studied in two models of immune-mediated inflammatory conditions in the CNS. These were: acute experimental allergic encephalomyelitis and aberrant delayed-type hypersensitivity lesions induced as a response to heat-killed bacillus Calmette-Guerin sequestrated behind the blood-brain barrier. In addition, a group of animals with a delayed- type hypersensitivity response was treated with dexamethasone to assess the effect of steroid treatment on T-cells and OX62+ cells in CNS lesions. Dendritic cells were present in many but not all lesions in acute experimental allergic encephalomyelitis and their numbers were small. In experimental allergic encephalomyelitis lesions, dendritic cells were found predominantly in perivascular cuffs, where they constituted approximately 2% of the total number of major histocompatibility complex class II+ cells. Some of these cells were also detected in the CNS parenchyma, close to the perivascular cuff. In contrast, dendritic cells were present in all delayed- type hypersensitivity lesions studied. Their number in delayed-type hypersensitivity lesions was significantly higher than in experimental allergic encephalomyelitis lesions. Numerous OX62+ cells were found, even in three-month-old lesions. Electron microscopy studies revealed that these cells were often in close contact with lymphocytes. There was no significant change in the density of OX62+ cells, IL2R+ cells and OX19+ T-cells in delayed-type hypersensitivity lesions after seven-day treatment with dexamethasone, although there was a considerable reduction in the number of CD45RA+ T-cells. The high numbers of dendritic cells found in the delayed- type hypersensitivity lesions may be important in contributing to the chronicity of the response. They may also initiate autoimmune responses to CNS antigens uncovered during bystander tissue damage which occurs as a consequence of aberrant delayed-type hypersensitivity responses.</p

Delayed-type hypersensitivity lesions in the central nervous system are prevented by inhibitors of matrix metalloproteinases

We have studied the effect of an inhibitor of matrix metalloproteinases, BB-1101, on a delayed-type hypersensitivity (DTH) response in the CNS. We used a recently described model in which heat-killed bacillus Calmette-Guerin (BCG) sequestered behind the blood-brain barrier (BBB) is targeted by a T-cell mediated response after subcutaneous injection of BCC (Matyszak and Perry, 1995). The DTH lesions are characterised by breakdown of the BBB, macrophage and lymphocyte infiltration and tissue damage including myelin loss. Treatment with BB-1101, which is not only a potent inhibitor of matrix metalloproteinases but also strongly inhibits TNF-α release, dramatically attenuated the CNS lesions. Breakdown of the BBB and the recruitment of T-cells into the site of the lesion were significantly reduced. There were many fewer inflammatory macrophages in DTH lesions than in comparable lesions from untreated animaIs. There was also significantly less myelin damage (assessed by staining with anti-MBP antibody). The DTH response in animals treated with dexamethasone was also reduced, but o a lesser degree. No significant effect was seen after administration of pentoxifylline, a phosphodiesterase inhibitor with effects including the inhibition of TNF-α production. Our results suggest that inhibitors of matrix metalloproteinases may be of considerable therapeutic benefit in neuroinflammatory diseases.</p

Stromal macrophages of the choroid plexus situated at an interface between the brain and peripheral immune system constitutively express major histocompatibility class II antigens

Using immunocytochemistry we have shown that there is a population of macrophages within the stroma of the choroid plexus of rats and mice which expresses high levels of major histocompatibility complex Class II antigens. In whole mount preparations of the choroid plexus, the morphology and regular distribution of these cells is similar to the Langerhans cells of the skin. These cells reside at an important interface between the central nervous system and the peripheral immune system and their possible role in immune-mediated diseases of the central nervous system is discussed.</p

Axonal damage in acute multiple sclerosis lesions

One of the histological hallmarks of early multiple sclerosis lesions is primary demyelination, with myelin destruction and relative sparing of axons. On the other hand, it is widely accepted that axonal loss occurs in, and is responsible for the permanent disability characterizing the later chronic progressive stage of the disease. In this study, we have used an antibody against amyloid precursor protein, known to be a sensitive marker of axonal damage in a number of other contexts, in immunocytochemical experiments on paraffin embedded multiple sclerosis lesions of varying ages in order to see at which stage of the disease axonal damage, in addition to demyelination, occurs and may thus contribute to the development of disability in patients. The results show the expression of amyloid precursor protein in damaged axons within acute multiple sclerosis lesions, and in the active borders of less acute lesions. This observation may have implications for the design and timing of therapeutic intervention, one of the most important aims of which must be the reduction of permanent disability.</p

The inflammatory response in the CNS

In recent years it has been recognized that cells of the mononuclear phagocyte lineage, macrophages and microglia, are a major component of gliosis. We review here studies on the kinetics of the myelomonocytic response to acute excitotoxin induced neuronal degeneration and following the injection of endotoxin (LPS) into the parenchyma of the central nervous system. These studies have shown that the kinetics of myelomonocytic recruitment to the parenchyma of the central nervous system is quite unlike that of other tissues; the polymorphonuclear cells are largely excluded and monocytes are only recruited after a delay of several days. The unusual nature of the inflammatory response in the central nervous system needs to be considered when drawing parallels with the acute inflammatory response in other tissues.</p