Pivotal advance: HMGB1 expression in active lesions of human and experimental multiple sclerosis

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. The underlying molecular mechanisms responsible for disease progression are still unclear. Herein, we demonstrate that high mobility group box chromosomal protein 1 (HMGB1), a DNA-binding protein with proinflammatory properties, is evident in active lesions of MS and experimental autoimmune encephalomyelitis (EAE) and that HMGB1 levels correlate with active inflammation. Furthermore, the expression of the innate HMGB1 receptors--receptor for advanced glycation end products, TLR2, and TLR4--was also highly increased in MS and rodent EAE. Additionally, in vitro activation of rodent CNS-derived microglia and bone marrow-derived macrophages demonstrated that microglia were equally as capable as macrophages of translocating HMGB1 following LPS/IFN-gamma stimulation. Significant expression of HMGB1 and its receptors on accumulating activated macrophages and resident microglia may thus provide a positive feedback loop that amplifies the inflammatory response during MS and EAE pathogenesis.     

TLR7 signaling exacerbates CNS autoimmunity through downregulation of Foxp3+ Treg cells

The innate Toll‐like receptor 7 (TLR7) detects infections by recognizing viral and bacterial single‐stranded RNA. In addition to pathogen‐derived RNA, immune cells expressing high levels of TLR7, such as B cells and dendritic cells (DCs), can be activated by self‐RNA. During myelin‐induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, TLR7 expression is increased within the central nervous system (CNS). To define the contribution of TLR7 to the development of EAE, we evaluated the course of the disease in C57BL/6‐Tlr7‐deficient mice compared with that in WT mice and found that TLR7‐deficient mice had decreased disease severity. This protection was associated with decreased myelin oligodendrocyte glycoprotein‐specific T‐cell activation by primed DCs, decreased circulating autoantibodies, attenuated inflammation within the CNS, and increased Foxp3⁺ regulatory T cells in the periphery and in the CNS. In conclusion, we show that TLR7 is involved in the maintenance of autoimmunity in the pathogenesis of EAE.     

Plasmacytoid DC promote priming of autoimmune Th17 cells and EAE

EAE, an animal model for MS, is a Th17 and Th1‐cell‐mediated autoimmune disease, but the mechanisms leading to priming of encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. To investigate the role of plasmacytoid DC (pDC) in the initiation of autoimmune Th17‐ and Th1‐cell responses and EAE, we depleted pDC with anti‐pDC Ag‐1 (anti‐PDCA1) mAb prior to immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG). pDC‐depleted mice developed less severe clinical and histopathological signs of EAE than control mice, which demonstrates a promoting role for pDC in the initiation of EAE. The levels of type I IFN were much lower in the sera from anti‐PDCA1‐treated mice. However, neutralization of type I IFN ameliorated the early phase of EAE but did not alter the severity of disease. Thus, only a minor part of the EAE‐promoting effect of pDC appears to be mediated by IFN‐α/β secretion. The numbers of MOG‐specific Th17 cells, but not Th1 cells, were lower in spleen from anti‐PDCA1‐treated mice compared with controls. In contrast, pDC depletion a week after MOG immunization resulted in more severe clinical signs of EAE. In conclusion, we demonstrate that pDC promote initiation of MOG‐induced Th17‐cell responses and EAE.     

Unexpected regulatory roles of TLR4 and TLR9 in experimental autoimmune encephalomyelitis

Innate immune mechanisms essential for priming encephalitogenic T cells in autoimmune neuroinflammation are poorly understood. Experimental autoimmune encephalomyelitis (EAE) is a IL-17-producing Th (Th17) cell-mediated autoimmune disease and an animal model of multiple sclerosis. To investigate how upstream TLR signals influence autoimmune T cell responses, we studied the role of individual TLR and MyD88, the common TLR adaptor molecule, in the initiation of innate and adaptive immune responses in EAE. Wild type (WT) C57BL/6, TLR-deficient and MyD88-deficient mice were immunized with myelin oligodendrocyte glycoprotein (MOG) in CFA. MyD88(-/-) mice were completely EAE resistant. Purified splenic myeloid DC (mDC) from MyD88(-/-) mice expressed much less IL-6 and IL-23, and serum and T cell IL-17 were absent. TLR4(-/-) and TLR9(-/-) mice surprisingly exhibited more severe EAE symptoms than WT mice. IL-6 and IL-23 expression by mDC and Th17 responses were higher in TLR4(-/-) mice, suggesting a regulatory role of TLR4 in priming Th17 cells. IL-6 expression by splenocytes was higher in TLR9(-/-) mice. Our data suggest that MyD88 mediates the induction of mDC IL-6 and IL-23 responses after MOG immunization, which in turn drives IL-17-producing encephalitogenic Th17 cell activation. Importantly, we demonstrate that TLR4 and TLR9 regulate disease severity in MOG-induced EAE.     

Differential macrophage/microglia activation in neocortical EAE lesions in the marmoset monkey

Recent studies revealed an important involvement of the cerebral cortex in multiple sclerosis (MS) patients. Cortical lesions in MS were reported to be less inflammatory and to show less structural damage than white matter lesions. Animal models reflecting the histopathological hallmarks of cortical demyelinated lesions in MS are sparse. Induction of experimental autoimmune encephalomyelitis (EAE) in the common marmoset has turned out to be an attractive non-human-primate model for MS. In the present study we investigated the presence and detailed cellular composition of cortical inflammatory demyelinating pathology in the common marmoset upon immunization with myelin oligodendrocyte glycoprotein (MOG). Extensive cortical demyelination reflecting the topographically distinct cortical lesion types in MS patients was revealed by immunohistochemistry for myelin basic protein (MBP). We explored the density of T- and B-lymphocytes, MHC-II expressing macrophages/microglia cells and early activated macrophages (MRP14) at perivascular and parenchymal lesions sites in neocortex and subcortical white matter. Despite a similar density of perivascular inflammatory infiltrates in the demyelinated neocortex, a considerable lower fraction of macrophages was found to express MRP14 in the neocortex indicating a different activation pattern in cortical compared with white matter lesions. Furthermore, cortical EAE lesions in marmoset monkeys revealed immunoglobulin leakage and complement component C9 deposition in intracortical but not subpial demyelination. Our findings indicate that the inflammatory response, especially macrophage and microglia activation, may be regulated differently in gray matter areas in primate brain.     

Ageing and recurrent episodes of neuroinflammation promote progressive experimental autoimmune encephalomyelitis in Biozzi ABH mice

Current therapies for multiple sclerosis (MS) reduce the frequency of relapses by modulating adaptive immune responses but fail to limit the irreversible neurodegeneration driving progressive disability. Experimental autoimmune encephalomyelitis (EAE) in Biozzi ABH mice recapitulates clinical features of MS including relapsing–remitting episodes and secondary‐progressive disability. To address the contribution of recurrent inflammatory events and ageing as factors that amplify progressive neurological disease, we examined EAE in 8‐ to 12‐week‐old and 12‐month‐old ABH mice. Compared with the relapsing–remitting (RREAE) and secondary progressive (SPEAE) EAE observed in young mice, old mice developed progressive disease from onset (PEAE) associated with pronounced axonal damage and increased numbers of CD3⁺ T cells and microglia/macrophages, but not B cells. Whereas the clinical neurological features of PEAE and SPEAE were comparable, the pathology was distinct. SPEAE was associated with significantly reduced perivascular infiltrates and T‐cell numbers in the central nervous system (CNS) compared with PEAE and the acute phase of RREAE. In contrast to perivascular infiltrates that declined during progression from RREAE into SPEAE, the numbers of microglia clusters remained constant. Similar to what is observed during MS, the microglia clusters emerging during EAE were associated with axonal damage and oligodendrocytes expressing heat‐shock protein B5, but not lymphocytes. Taken together, our data reveal that the course of EAE is dependent on the age of the mice. Younger mice show a relapsing–remitting phase followed by progressive disease, whereas old mice immediately show progression. This indicates that recurrent episodes of inflammation in the CNS, as well as age, contribute to progressive neurological disease.     

Critical role for PDE4 subfamilies in the development of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis exhibiting neuroinflammation, axonal damage and demyelination, further characterized by T- and B-cell responses to myelin oligodendrocyte glycoprotein. Pharmacological manipulation of phosphodiesterases (PDEs) provokes profound anti-inflammatory responses through modulation of cAMP levels. The PDE4B subfamily has been related to the inflammatory immune response in mice and PDE4 inhibition produces amelioration of the clinical signs and delayed onset in the EAE model. Analyses of the expression of the mRNA coding for PDE4B splice variants revealed an upregulation of PDE4B2 in the brainstem and spinal cord of EAE mice which correlated with forkhead box P3 (FoxP3) and transforming growth factor beta (TGF-β) mRNAs expression in a score-dependent manner. The increase observed for the PDE4B protein was mainly found in antigen-presenting cells (APCs) such as dendritic cells and microglia/macrophages, in areas with high cellular infiltration. Unexpectedly, PDE4B^−/− mice showed an earlier onset of the disease compared to wildtype mice. The results point to a possible role of the PDE4B enzyme and in particular the PDE4B2 splice variant during EAE pathogenesis, probably by modulating cAMP levels in APCs, consequently influencing the cytokine environment important for T-cell differentiation.     

Lack of adiponectin leads to increased lymphocyte activation and increased disease severity in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a presumed autoimmune disease directed against central nervous system (CNS) myelin, in which diet and obesity are implicated as risk factors. Immune responses can be influenced by molecules produced by fat cells, called adipokines. Adiponectin is an adipokine with anti‐inflammatory effects. We tested the hypothesis that adiponectin has a protective role in the EAE model for MS, that can be induced by immunization with myelin antigens or transfer of myelin‐specific T lymphocytes. Adiponectin deficient (ADPKO) mice developed worse EAE with greater CNS inflammation, demyelination, and axon injury. Lymphocytes from myelin‐immunized ADPKO mice proliferated more, produced higher amounts of IFN‐γ, IL‐17, TNF‐α, IL‐6, and transferred more severe EAE than wild type (WT) lymphocytes. At EAE peak, the spleen and CNS of ADPKO had fewer regulatory T (Treg) cells than WT mice and during EAE recovery, Foxp3, IL‐10 and TGF‐β expression levels in the CNS were reduced in ADPKO compared with WT mice. Treatment with globular adiponectin in vivo ameliorated EAE, and was associated with an increase in Treg cells. These data indicate that adiponectin is an important regulator of T‐cell functions during EAE, suggesting a new avenue of investigation for MS treatment.     

Endotoxin- and ATP-neutralizing activity of alkaline phosphatase as a strategy to limit neuroinflammation

Background

Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches.

Methods

Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects.

Results

AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS.

Conclusions

Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.

     

Alpha‐methylacyl‐CoA racemase deletion has mutually counteracting effects on T‐cell responses,associated with unchanged course of EAE

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha‐methylacyl‐CoA racemase (AMACR) converts R‐configuration branched fatty acids into the S‐configuration, thereby preparing them for β‐oxidation. We observed a significant, disease‐dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T‐cell polarization was switched toward a pathogenic state involving the production of more IFN‐γ and IL‐17, but less IL‐4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.     

Fc receptors are critical for autoimmune inflammatory damage to the central nervous system in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is simulated by various forms of experimental autoimmune encephalomyelitis, in which T cells, antibodies, cytokines and complementary factors interact with the central nervous system (CNS) myelin proteins and lead to inflammatory damage. We investigated the role of Fc receptors (FcRs), which link the cellular and humoral branches of the immune system, in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), using two different FcRgamma knockout DBA/1 mice. The first knockout were the FcRgamma chain-deficient mice, which lack FcgammaRI, FcgammaRIII and Fc(epsilon)RI, while the second knockout mice lack only FcgammaRII. The lack of FcgammaRII enhanced the disease susceptibility with associated increased CNS demyelination. While FcRgamma+/+ DBA/1 mice also developed pronounced CNS infiltration and myelin destruction, FcRgamma-/- littermates were protected despite initial peripheral autoimmune responses to MOG. In vitro analyses revealed equivalent potentials of fluid phase phagocytosis of myelin and MOG in bone-marrow macrophages derived from both FcRgamma+/+ and FcRgamma-/- mice, while MOG-immunoglobulin (Ig)G immune complexes were only internalized by FcRgamma+/+ macrophages. This was associated with cellular activation in FcRgamma+/+ but not FcRgamma-/- macrophages, as assessed by the activation of intracellular mitogen activated protein (MAP)-kinase signalling elements. We propose that protection from EAE in FcRgamma-deficient mice is due to the inefficient antigen processing/presentation of myelin proteins during the induction of secondary immune responses locally in the CNS, which leads to demyelination. This demonstrates the importance of FcR in the promotion of autoimmune inflammation of the CNS and highlights the therapeutic possibility of treatment of MS with FcR-directed modalities.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

Inhibition of glial cell activation ameliorates the severity of experimental autoimmune encephalomyelitis

Activated microglia and astrocytes have been implicated in the course of multiple sclerosis (MS) and its animal model: experimental autoimmune encephalomyelitis (EAE). MW01-5-188WH is a novel drug that selectively inhibits glial activation in the central nervous system (CNS). We report here that MW01-5-188WH is effective to ameliorate the severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Daily oral administration of MW01-5-188WH at 5mg/kg body weight reduced the clinical scores of EAE mice while having no influence on the disease incidence or animal mortality. Pathological examination revealed reduced numbers of microglia and astrocytes in the spinal cord of MW01-5-188WH-treated EAE mice. Moreover, MW01-5-188WH suppressed the release of key chemokines, which are involved in MS pathology, from cultured microglia and astrocytes. Taken together, our results indicate that treatments that suppress the activation of microglia and astrocytes should be pursued in future research for their potential as avenues for the treatment of MS.     

Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

Macrophage inflammatory protein (MIP)-1alpha is a chemokine that is associated with Th1 cytokine responses. Expression and antibody blocking studies have implicated MIP-1alpha in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). We examined the role of MIP-1alpha and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild-type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential chemokine expression patterns represent differences in disease mechanism that underlie various models of EAE, and possibly distinct patterns of pathology seen in MS.     

Probenecid Application Prevents Clinical Symptoms and Inflammation in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Neurological impairments are caused by axonal damage due to demyelination and neuroinflammation within the central nervous system. T cells mediate the neuroinflammation. The activation of T cells is induced by the release of adenosine triphosphate and involves purinergic receptors as well as pannexin (Panx) proteins. As Panx1 is expressed on T cells, we here propose that application of probenecid, a known Panx inhibitor, will prevent the onset of clinical symptoms in a mouse model of MS, the experimental autoimmune encephalomyelitis (EAE) model. EAE-induced mice received daily injections of probenecid. Disease scores, T cell numbers, and microglia activation were compared between experimental groups. Probenecid treatment resulted in lower disease scores as compared to EAE animals. Probenecid-treated animals also displayed fewer inflammatory lesions. Microglia activation was not altered by treatment. In conclusion, probenecid prevented the onset of EAE.     

Complementation between specific HLA-DR and HLA-DQ genes in transgenic mice determines susceptibility to experimental autoimmune encephalomyelitis

To investigate the contribution of human leukocyte antigen (HLA) class II molecules in susceptibility to inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in transgenic (tg) mice expressing the HLA-DR3, HLA-DQ8 and HLA-DQ6 molecules in the absence of endogenous class II (Ab(o)). Following immunization with mouse myelin, HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed inflammatory lesions and demyelination in the central nervous system with mild to moderate clinical symptoms of EAE. HLA-DQ8 and HLA-DQ6 tg mice elicited weak T-cell proliferative responses and did not develop clinical symptoms of EAE. HLA-DR3/DQ6 double tg mice immunized with mouse myelin experienced clinical disease similar to the single tg HLA-DR3 tg mice, indicating that expression of DQ6 in this line had no effect on disease. In contrast, HLA-DR3/DQ8 double tg mice developed severe inflammatory lesions and clinical disease in response to immunization with mouse myelin. Our data suggest that in the presence of two susceptible class II alleles, namely HLA-DR3 and DQ8, there is additional selection and expansion of potential autoreactive T cells, resulting in enhanced severity of disease.     

MHC gene related effects on microglia and macrophages in experimental autoimmune encephalomyelitis determine the extent of axonal injury

Myelin-oligodendrocyte-glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in rats is a chronic inflammatory demyelinating disease of the central nervous system (CNS) strongly mimicking multiple sclerosis (MS). We determined the involvement of macrophages and microglia in the lesions of MOG-EAE in relation to different major histocompatibility complex (MHC, RT1 in rat) haplotypes. We used intra-RT1 recombinant rat strains with recombinations between the RT1a and RT1u haplotypes on the disease permissive LEW non-MHC genome. Activated microglia and macrophages were identified morphologically and by expression of ED1 and allograft inhibitory factor-1 (AIF-1), and differentiated by their morphological phenotype. White matter lesions contained more macrophages and less microglia compared to grey matter lesions. Similarly active lesions were mainly infiltrated by macrophages, while microglia were abundant in inactive demyelinated plaques. In addition, we found a highly significant genetic association between a macrophage or microglia dominated lesional phenotype, which was independent from location and activity of the lesions. This was not only the case in demyelinating plaques of chronic EAE, but also in purely inflammatory lesions of acute passive transfer EAE. Rat strains with an u-haplotype in both the Class II and the telomeric non-classical Class I region revealed inflammatory and demyelinating lesions, which were dominated by activated microglia. The a-haplotype in any of these regions was associated with macrophage dominated lesions. A comparison of lesions, exactly matched for stages of demyelinating activity in these different rat strains, showed that in spite of a similar extent of demyelination, axonal injury was significantly less in microglia compared to macrophage dominated lesions. Thus, our studies document a genetic influence of the MHC-region on the relative contribution of macrophages versus microglia in the pathogenesis of EAE.     

Microglial MHC class II is dispensable for experimental autoimmune encephalomyelitis and cuprizone‐induced demyelination

Microglia are resident immune cells in the CNS, strategically positioned to clear dead cells and debris, and orchestrate CNS inflammation and immune defense. In steady state, these macrophages lack MHC class II (MHCII) expression, but microglia activation can be associated with MHCII induction. Whether microglial MHCII serves antigen presentation for critical local T‐cell restimulation in CNS auto‐immune disorders or modulates microglial signaling output remains under debate. To probe for such scenarios, we generated mice harboring an MHCII deficiency in microglia, but not peripheral myeloid cells. Using the CX₃CR1^CreER‐based approach we report that microglial antigen presentation is obsolete for the establishment of EAE, with disease onset, progression, and severity unaltered in mutant mice. Antigen presentation‐independent roles of microglial MHCII were explored using a demyelination model induced by the copper chelator cuprizone. Absence of microglial I‐A^b did not affect the extent of these chemically induced white matter alterations, nor did it affect microglial proliferation or gene expression associated with locally restricted de‐ and remyelination.     

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.