Microglia are more susceptible than macrophages to apoptosis in the central nervous system in experimental autoimmune Encephalomyelitis through a mechanism not involving Fas (CD95)

Morphological studies have shown that macrophages and microglia undergo apoptosis in the central nervous system (CNS) in acute experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. To assess the relative levels of macrophage and microglial apoptosis, and the molecular mechanisms involved in this process, we used three-colour flow cytometry to identify CD45lowCD11b/c+ microglial cells and CD45highCD11b/c+ macrophages in the inflammatory cells isolated from the spinal cords of Lewis rats 13 days after immunization with myelin basic protein (MBP) and complete Freund's adjuvant. Simultaneously, we analyzed the DNA content of these cell populations to assess the proportions of cells undergoing apoptosis and in different stages of the cell cycle or examined their expression of three apoptosis-regulating proteins, i.e. Fas (CD95), Fas ligand (FasL) and Bcl-2. Microglia were highly vulnerable to apoptosis and were over-represented in the apoptotic population. Macrophages were less susceptible to apoptosis than microglia and underwent mitosis more frequently than microglia. The different susceptibilities of microglia and macrophages to apoptosis did not appear to be due to variations in Fas, FasL or Bcl-2 expression, as the proportions of microglia and macrophages expressing these proteins were similar, and were relatively high. Furthermore, in contrast to T cell apoptosis, apoptosis of microglia/macrophages did not occur more frequently in cells expressing Fas or FasL, or less frequently in cells expressing Bcl-2. These results indicate that the apoptosis of microglia and CNS macrophages in EAE is not mediated through the Fas pathway, and that Bcl-2 expression does not protect them from apoptosis. Expression of FasL by macrophages and microglia may contribute to the pathogenesis and immunoregulation of EAE through interactions with Fas+ oligodendrocytes and Fas+ T cells. The high level of microglial apoptosis in EAE indicates that microglial apoptosis may be an important homeostatic mechanism for controlling the number of microglia in the CNS following microglial activation and proliferation

Cyclosporin A Treatment Modulates Cytokine mRNA Expression by Inflammatory Cells Extracted from the Spinal Cord of Rats with Experimental Autoimmune Encephalomyelitis Induced by Inoculation with Myelin Basic Protein

In Lewis rats, treatment with high doses of cyclosporin A (CsA) suppresses clinical signs of experimental autoimmune encephalomyelitis (EAE), although disease occurs when treatment is ceased. Treatment with low doses of CsA causes EAE to take a chronic relapsing course. We have previously shown that CsA treatment causes a decline in the number of T cells and increased inflammatory cell apoptosis in the spinal cord. The present study was undertaken to assess whether CsA therapy also modulates cytokine mRNA expression by inflammatory cells in the spinal cord of rats with EAE, looking for changes that might contribute to the observed effects of CsA on the course of EAE. EAE was induced in Lewis rats by inoculation with myelin basic protein and adjuvants. At the peak of neurological signs, on day 14 after inoculation, rats were given a single intraperitoneal injection of saline, or CsA at a dose of 8, 16, 32 or 64 mg/kg. The next day, rats were sacrificed, the spinal cords removed, inflammatory cells were extracted from the cords, and mRNA isolated from these cells. Expression of cytokine mRNA was assessed by semi-quantitative reverse transcription polymerase chain reaction (PCR) and by quantitative real-time PCR. With both techniques, we found that CsA suppressed the expression of interferon-gamma mRNA and interleukin-2 (IL-2) mRNA. With real-time PCR, we found that CsA caused significantly increased expression of transforming growth factor-beta mRNA. With the different techniques, we observed no consistent pattern of alteration of expression of interleukin-10 or interleukin-4 mRNA. It is possible that these changes in cytokine mRNA expression contribute to the modulation of the clinical course of EAE that is produced by CsA treatment

Effects of Cyclosporin A Treatment on Clinical Course and Inflammatory Cell Apoptosis in Experimental Autoimmune Encephalomyelitis Induced in Lewis Rats by Inoculation with Myelin Basic Protein

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by inoculation with myelin basic protein (MBP) and adjuvants. Rats were treated with second daily injections of saline or cyclosporin A (CsA) from the day of inoculation. Saline-treated rats had an acute episode of disease followed by clinical recovery. Rats treated with CsA 16 or 32 mg/kg had minimal signs of EAE at the usual time after inoculation, but developed signs of disease after treatment was ceased. Rats treated with CsA 8 mg/kg had a delayed first episode of disease and then developed a relapsing or a chronic persistent course of disease. CsA 4 mg/kg delayed the onset of disease. To study the effects of CsA on the inflammatory infiltrate, cells were extracted from the spinal cords of rats with EAE, 16 h after a single injection of CsA or saline. Extracted cells were labelled with antibodies to T cells, CD11b/c (macrophages/microglia), CD95 (Fas) and Fas ligand. CsA 4 mg/kg did not alter the composition of the inflammatory infiltrate. Treatment with higher single doses of CsA caused a dose-dependent decline in the percentage of T cell receptor (TCR)alpha beta+ cells in the inflammatory infiltrate. All doses of CsA caused a significant increase in the number and percentage of cells that were apoptotic. CsA treatment caused an increase in the percentages of CD5+ and TCR alpha beta+ cells that were apoptotic. There was a decline in the percentage of apoptotic T cells that were V beta 8.2+, compared to the percentage of non-apoptotic T cells that were V beta 8.2+, in CsA treated rats compared to saline-treated controls. This suggests that, while CsA treatment caused a non-specific increase in the overall level of T cell apoptosis in the spinal cord, it abrogated the selective apoptosis of V beta 8.2+ encephalitogenic T cells that normally occurs during spontaneous recovery from acute EAE

Antigen-Specific Down-Regulation of Myelin Basic Protein-Reactive T Cells During Spontaneous Recovery From Experimental Autoimmune Encephalomyelitis: Further Evidence of Apoptotic Deletion of Autoreactive T Cells in the Central Nervous System

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by the i.v. injection of 107 cloned V beta 8.2+ T cells specific for the 72-89 peptide of guinea pig myelin basic protein (MBP). Some animals were injected simultaneously with 107 cloned T cells specific for ovalbumin (OVA). Lymphocytes were isolated from the spinal cord and from the peripheral lymphoid organs of these rats and the frequencies of MBP-peptide-specific or OVA-specific proliferating cells were estimated by limiting dilution analysis at different times after cell transfer. The frequencies of cells specific for MBP_72-89 or OVA in the spinal cord were highest 5 days after cell transfer (MBP_72-89, 1 in 1149; OVA, 1 in 1116). On day 7, when the rats were recovering, the frequency of cells specific for MBP_72-89 in the spinal cord fell dramatically t

Macrophage apoptosis in the central nervous system in experimental autoimmune encephalomyelitis

Using light and electron microscopy, we have demonstrated that macrophage apoptosis (programmed cell death) occurs in the central nervous system (CNS) in Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) and chronic relapsing EAE. Apoptotic macrophages were identified by the presence of an apoptotic nucleus in a cell with cytoplasm containing myelin debris but no intermediate filaments. They were found in the meninges, perivascular spaces and in the parenchyma of the white and grey matter of the spinal cord. In acute EAE the apoptotic macrophages were most frequently seen at the time of maximal neurological signs and during the early stages of clinical recovery. Several possible mechanisms may be responsible for the macrophage apoptosis: the release or withdrawal of cytokines; T-cell cytotoxicity; the effect of activated macrophage products, such as nitric oxide; and a direct effect of endogenous glucocorticoids. Macrophage apoptosis, together with the T-cell apoptosis we have previously described in the CNS in EAE, may contribute to the down-regulation of this autoimmune disease

Conduction abnormalities are restricted to the central nervous system in experimental autoimmune encephalomyelitis induced by inoculation with proteolipid protein but not with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) and can be induced by inoculation of animals with homogenized CNS tissue or highly purified myelin proteins such as myelin basic protein (MBP) or proteolipid protein (PLP). It is widely studied as a possible animal model of multiple sclerosis. We performed the present neurophysiological study to define the location of nerve conduction abnormalities in EAE induced by immunization with PLP (PLP-EAE) and in EAE induced by immunization with MBP (MBP-EAE) in the Lewis rat. In rats with tail weakness due to acute PLP-EAE, conduction was normal in the spinal nerve roots and peripheral nerves but there was evidence of conduction block in a high proportion of the fibres in the dorsal columns of the lumbosacral spinal cord. In contrast, in acute MBP-EAE, there was conduction block in a high proportion of fibres in the sacral dorsal and ventral roots of the peripheral nervous system (PNS) and in the dorsal columns of the lumbosacral spinal cord. The distribution of nerve conduction abnormalities is consistent with previous histological studies showing that inflammation and primary demyelination are restricted to the CNS in PLP-EAE, but are present in the CNS and in the spinal roots of the PNS in MBP-EAE. The restriction of functional abnormalities to the CNS in PLP-EAE but not in MBP-EAE may have implications for the human inflammatory demyelinating diseases, including multiple sclerosis

Apoptosis of αβ T lymphocytes in the nervous system in experimental autoimmune encephalomyelitis: Its possible implications for recovery and acquired tolerance

We have recently shown that apoptosis, an active process of cellular self-destruction, occurs in the central nervous system in Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Conventional light and electron microscopic studies suggested that some of the apoptotic cells were oligodendrocytes and that others were hematogenous mononuclear cells. To determine whether any of the apoptotic cells were T lymphocytes, we used the technique of pre-embedding immunolabelling which allows sufficient preservation of the ultrastructure to permit recognition of apoptotic changes while at the same time preserving surface antigens so that the identity of the apoptotic cells can be determined by immunocytochemistry. Light microscopic immunocytochemistry using the mono-clonal antibodies OX-34 (CD2) and R73 (alpha beta T-cell receptor) revealed that 10% of the CD2+ cells and 5% of the alpha beta T lymphocytes in the parenchyma of the spinal cord were dying by apoptosis. The presence of apoptotic alpha beta T cells was confirmed by electron microscopy. About half of all the apoptotic cells within the spinal cord were labelled by these antibodies. It is possible that some of the unlabelled apoptotic cells were also T lymphocytes but that others were glial cells such as oligodendrocytes. One possible interpretation of this T-cell apoptosis is that it represents activation-induced cell death, which has recently been shown to provide a mechanism of clonal elimination of mature as well as immature autoreactive T cells. Another possible interpretation is that it is a result of corticosterone released during the course of EAE. The apoptotic elimination of target-antigen-specific lymphocytes within the target organ in this autoimmune disease may contribute to the subsidence of inflammation and, if ongoing, to the development of tolerance

Lack Of Neurological Abnormalities In Lewis Rats With Experimental Chronic Serum Sickness

Serum sickness in man may occur after treatment with foreign proteins such as tetanus or diphtheria antisera, and in some patients leads to neurological complications such as neuropathy or encephalomyelitis. Many of the effects of serum sickness are associated with the deposition of antigen-antibody complexes in the tissues. Chronic serum sickness in the rabbit has previously been shown to cause perivascular inflammation and demyelination in the nervous system. We induced chronic serum sickness in the Lewis rat by daily intraperitoneal injections of bovine serum albumin (BSA) in male rats that had previously received footpad inoculations of BSA. Two animals died of anaphylaxis and 15 were observed for periods of 39 to 142 days. Three animals injected with 3 mg or 4 mg/day of BSA, and 6 animals injected with up to 16 mg/day of BSA had no clinical abnormalities when sacrificed. Six animals were injected with 36 to 40 mg BSA/day and, at the time of sacrifice, were lethargic and had ruffled fur, but no neurological signs. In these animals, the production of chronic serum sickness was confirmed by the presence of immune complex deposits in the kidneys. In the nervous system, there was no evidence of inflammatory cell infiltration either in the parenchyma or the vessel walls. Immunofluorescence studies identified deposits of immunoglobulin in the choroid plexus of chronic serum sickness rats but not in controls. Staining with antibodies to immunoglobulin, complement and BSA showed marked staining of blood vessels of the nerve roots of the animals with chronic serum sickness. There was also some minor immunofluorescent staining with these markers in the blood vessels of the nerve roots of control animals, but this was always less than in chronic serum sickness animals

Serial measurements of phosphorylated neurofilament-heavy in the serum of subjects with amyotrophic lateral sclerosis

There is a need for a blood biomarker of disease activity in ALS. This marker needs to measure the loss of motor neurones. Phosphorylated neurofilament heavy chain (pNfH) in the serum is a biomarker of axonal injury. Previous studies have found that levels of pNfH are elevated in ALS. We have performed a serial study of pNfH levels in 98 subjects from our ALS clinic. There was significant elevation of levels of pNfH in subjects with ALS compared to controls, although there was considerable variability. In studies of individuals who had two or more serial samples, we found that the levels of pNfH increased over time in the early stage of disease. Levels were low in subjects with long survival. The rate of rise of pNfH was inversely correlated with survival. We suggest that the initial level of pNfH is a marker of disease severity and that changes in pNfH levels are markers of disease progression