Characterizing primary human microglia: A comparative study with myeloid subsets and culture models.

The biology of microglia has become subject to intense study, as they are widely recognized as crucial determinants of normal and pathologic brain functioning. While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro- and anti-inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro- (LPS or poly I:C) and anti-inflammatory (IL-4 or dexamethasone) stimuli. Interestingly, pro-inflammatory responses differed between microglia and monocyte-derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. GLIA 2016;64:1857-1868.

Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigen-specific T cells.

Previous experiments from this laboratory have shown that Lewis rats were protected from experimental allergic encephalomyelitis (EAE) induced by the injection of myelin basic protein (MBP) in Freund's complete adjuvant if they were treated with the encephalitogenic peptide of MBP covalently linked to mouse anti-rat immunoglobulin (Ig) D. It was suggested that this protection developed because the antibody-peptide conjugate targeted the peptide to B cells and that this mode of presentation induced a Th2-like T cell response that controlled the concomitant encephalitogenic Th1 reaction to the autoantigen. The current experiments were carried out to test this hypothesis and to examine the alternative explanation for the protective effect of the conjugate pretreatment, namely that it induced a state of nonresponsiveness in the autoantigenspecific T cells. It was shown that EAE induction was suppressed in Lewis rats when the antibody-peptide conjugate was injected intravenously 14 and 7 d before immunization with MBP in adjuvant, but that anti-MBP antibody titers were at least as high in these animals as in controls that were not pretreated with the conjugate before immunization. Lymph node cells from these pretreated animals, while proliferating in vitro to MBP as vigorously as those from controls, produced less interferon gamma and were very inferior in their ability to transfer disease after this in vitro activation. In contrast, these same lymph node cells from protected rats generated markedly increased levels of messenger RNA for interleukin (IL)-4 and IL-13. When these in vitro experiments were repeated using the encephalitogenic peptide rather than MBP as the stimulus, the proliferative response of lymph node cells from pretreated donors was less than that from controls but was still readily detectable in the majority of experiments. Furthermore, the cytokine expression induced by the peptide was similar to that elicited by whole MBP. While these results support the original hypothesis that the anti-IgD-peptide conjugate pretreatment protected rats from EAE by inducing a Th2-type cytokine response, a totally unexpected finding was that this pretreatment greatly reduced the level of leukocyte infiltration into the central nervous system. This result provides a direct explanation for the protective effect of the pretreatment, but it raises questions regarding migratory and homing patterns of leukocytes activated by different immunological stimuli.

Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages.

Almost 50% of the cells infiltrating the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) are macrophages (M psi). To investigate the role of the M psi in the pathogenesis of EAE, we eliminated M psi by means of mannosylated liposomes containing dichloromethylene diphosphonate (Cl2MDP). Cl2MDP-containing liposomes injected intravenously eliminate M psi in spleen and liver. Incorporation of mannose into the lipid layers enables the liposomes to pass the blood-brain barrier (BBB). Injections of Cl2MDP-containing mannose liposomes intravenously shortly before the appearance of clinical signs, markedly suppressed the expression of clinical signs of EAE. This suppression was accompanied by a marked reduction of infiltrated M psi in the CNS. Cl2MDP-containing liposomes without mannose incorporated had no effect. Cl2MDP-containing mannosylated liposomes had no effect on plasma corticosterone levels compared with injections of saline; thus, the suppression of expression of EAE was not corticosterone mediated. These results show that the M psi within the CNS play an important role in the pathogenesis of EAE.

Neuropathological correlates of parkinsonian disorders in a large Dutch autopsy series.

The clinical diagnosis in patients with parkinsonian disorders can be challenging, and a definite diagnosis requires neuropathological confirmation. The aim of this study was to examine whether a clinical diagnosis of Parkinson's disease (PD) and atypical parkinsonian disorders predict the presence of Lewy pathology (LP) and concomitant neuropathological lesions.We included 293 donors with a history of parkinsonism without dementia at disease onset, collected by the Netherlands Brain Bank (NBB) from 1989 to 2015. We retrospectively categorized donors according the International Parkinson and Movement Disorder Society clinical diagnostic criteria for PD (MDS-PD criteria) as 'not PD', 'probable PD' or 'established PD'. We compared the final clinical diagnosis to presence of neuropathological lesions as defined by BrainNet Europe and National Institute on Aging - Alzheimer's Association guidelines.LP was present in 150 out of 176 donors (85%) with a clinical diagnosis of PD, in 8 out of 101 donors (8%) with atypical parkinsonian disorders and in 4 out of 16 donors (25%) without a definite clinical diagnosis. Independent from age at death, stages of amyloid-ß, but not neurofibrillary tau or neuritic plaques, were higher in donors with LP compared to other types of pathology (p = 0.009). The MDS-PD criteria at a certainty level of 'probable PD' predicted presence of LP with a diagnostic accuracy of 89.3%. Among donors with LP, 'established PD' donors showed similar Braak α-synuclein stages and stages of amyloid-ß, neurofibrillary tau and neuritic plaques compared to 'not PD' or 'probable PD' donors.In conclusion, both a clinical diagnosis of PD as well as MDS-PD criteria accurately predicted presence of LP in NBB donors. LP was associated with more widespread amyloid-ß pathology, suggesting a link between amyloid-ß accumulation and LP formation.

Gene expression analysis in the human hypothalamus in depression by laser microdissection and real-time PCR: the presence of multiple receptor imbalances.

Hyperactivity of corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus (PVN) of the hypothalamus is a prominent feature in depression and may be important in the etiology of this disease. The activity of the CRF neurons in the stress response is modulated by a number of factors that stimulate or inhibit CRF expression, including (1) corticosteroid receptors and their chaperones, heat shock proteins 70 and 90, (2) sex hormone receptors, (3) CRF receptors 1 (CRFR1) and 2, (4) cytokines interleukin 1-beta and tumor necrosis factor-alpha, (5) neuropeptides and receptors, vasopressin (AVP), AVP receptor 1a (AVPR1A) and oxytocin and (6) transcription factor cAMP-response element-binding protein. We hypothesized that, in depression, the transcript levels of those genes that are involved in the activation of the hypothalamo-pituitary-adrenal (HPA) axis are upregulated, whereas the transcript levels of the genes involved in the inhibition of the HPA axis are downregulated. We performed laser microdissection and real-time PCR in the PVN and as a control in the supraoptic nucleus. Snap-frozen post-mortem hypothalami of seven depressed and seven matched controls were used. We found significantly increased CRF mRNA levels in the PVN of the depressed patients. This was accompanied by a significantly increased expression of four genes that are involved in the activation of CRF neurons, that is, CRFR1, estrogen receptor-alpha, AVPR1A and mineralocorticoid receptor, while the expression of the androgen receptor mRNA involved in the inhibition of CRF neurons was decreased significantly. These findings raise the possibility that a disturbed balance in the production of receptors may contribute to the activation of the HPA axis in depression.

Stress and hypothalamic-pituitary-adrenal axis function in experimental autoimmune encephalomyelitis and multiple sclerosis - a review.

Multiple sclerosis (MS) is an inflammatory and degenerative disease of the CNS with an assumed autoimmune-mediated pathogenesis. Stressful life events have been hypothesized as potential triggers of disease exacerbation. Animal studies using experimental autoimmune encephalomyelitis (EAE), as a model for MS, suggest that decreased hypothalamic-pituitary-adrenal (HPA) function may play a role in the increased susceptibility and severity of the disease. Histopathological studies of the hypothalamus point to disturbances in corticotropin-releasing hormone (CRH) regulation as a result of MS lesions in this area. Functional endocrine tests (e.g., the combined Dexamethasone-CRH test) showed a disturbed negative feedback after steroid application in MS patients. Hyper- and hypoactivity of the HPA axis, have been described to be associated with more severe courses. This paper presents an overview of the evidence for a role of HPA dysfunction in EAE and MS based on stress-experimental studies.

Hypothalamic lesions in multiple sclerosis.

Demyelinating lesions of fiber bundles in and adjacent to the hypothalamus (i.e. the fornix. anterior commissure, internal capsule, and optic system) may be the basis for autonomic and endocrine alterations in multiple sclerosis (MS) patients. Therefore we investigated the presence and immunological activity of lesions in hypothalamic fiber bundles of 17 MS patients and 14 controls. In the MS group, 16 of 17 patients showed demyelinated lesions. The incidence of active lesions was high (60%) and outnumbered chronic inactive lesions in the internal capsule (p = 0.005). In 4 of 17 MS patients, axonal damage was observed and in 3 of 17 MS patients grey matter lesions were apparent. Duration of MS was inversely related to the active hypothalamic MS lesion score (r = -0.72, p = 0.001). Since comparison of hypothalamic lesions with MS lesions in other areas of the brain in the same patients (n = 7) showed a great similarity both as stage and appearance was concerned, this negative relation in all likelihood reflects the clinical consequences of high disease activity throughout the whole brain. In controls no demyelinating lesions were seen but in 11 control cases HLA expression was observed that was lower than that present in MS patients (p = 0.02). In the median eminence region that lacks a blood-brain barrier, all controls showed a strong HLA expression around the blood vessels. We conclude that systematic pathological investigation of the hypothalamus in MS patients reveals an unexpected high incidence of active lesions that may impact on hypothalamic functioning.

The role of macrophages in demyelination.

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS). In particular in the CsA-induced chronic relapsing form (CREAE), pronounced demyelination occurs, in temporal association with relapses. It is still a matter of discussion which cell type ultimately is responsible for the actual process of demyelination. Macrophages, cytotoxic T lymphocytes and also astrocytes are possible candidates. In this short overview, the role of macrophages in the pathogenesis of EAE is discussed. It is shown that in particular, newly recruited macrophages play a crucial role in the generation of clinical signs. Possible mechanisms by which macrophages are involved in the pathogenesis of demyelinating diseases are presented.

Reactive oxygen species are involved in the pathogenesis of experimental allergic encephalomyelitis in Lewis rats.

During experimental allergic encephalomyelitis (EAE), both blood-borne macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions in the central nervous system (CNS), resulting in the neurological deficits common to EAE. Both cell types can produce multiple mediators of tissue damage, among which are the reactive oxygen species (ROS). In this study we show that macrophages and microglial cells, isolated from the CNS of Lewis rats with clinical signs of EAE, exhibited significantly elevated spontaneous and phorbol myristate acetate (PMA)-inducible levels of ROS compared to similar cells isolated from healthy controls, sham (complete Freund's adjuvant, CFA)-immunized rats as well as rats sacrificed before the manifestation of clinical signs of EAE. However, during clinical EAE, peripheral blood mononuclear cells (PBMC) did not show increased spontaneous nor PMA-inducible ROS production compared to controls. In vivo treatment of EAE with catalase, which scavenges the ROS H2O2, markedly suppressed the severity of the disease as compared to sham (albumin)-treated controls. In contrast, superoxide dismutase had no effect on clinical signs. Our studies point at a putative functional role for ROS, and in particular H2O2, in the pathogenesis of EAE.

Elimination of blood-derived macrophages inhibits the release of interleukin-1 and the entry of leukocytes into the cerebrospinal fluid in experimental pneumococcal meningitis.

Parameters of inflammation during pneumococcal meningitis were determined in rabbits after monocyte elimination by dichloromethylene diphosphonate (Cl(2)MDP)-containing mannosylated liposomes in comparison with untreated controls. Monocyte depletion reduced the migration of white blood cells into the cerebrospinal fluid (CSF) (medians: 42 versus 2146/mm3 at 18 h, 323 versus 7413/mm3 at 24 h p.i., p < 0.01). CSF IL-1beta concentrations were lower in depleted animals (379 versus 3282 pg/ml, 24 h p.i., p < 0.01), whereas TNF-alpha concentrations were not different. Monocyte-depleted animals lost body temperature during the experiment carried out in anaesthesia (p = 0.01) indicating that macrophages are necessary for thermogenesis during meningitis.

Reduction of meningeal macrophages does not decrease migration of granulocytes into the CSF and brain parenchyma in experimental pneumococcal meningitis.

Leukocyte infiltration of the CSF and brain parenchyma and other parameters of inflammation during pneumococcal meningitis were investigated after reduction of meningeal macrophages in rabbits by intracisternal injection of dichloromethylene-diphosphonate (Cl2MDP)-containing liposomes. Macrophages in the meninges were reduced, in median, by approximately 77% after three intrathecal injections of 100 microl of liposomes containing Cl2MDP at 12 h intervals. Production of the cytokines interleukin-1 and tumor necrosis factor-alpha as well as infiltration of the CSF and nervous tissue by leukocytes was not significantly altered in infected animals after treatment with Cl2MDP-containing liposomes. The median CSF concentration of neuron specific enolase (NSE) as a parameter of neuronal damage was higher in infected Cl2MDP-treated animals (median [median (25th/75th percentiles): 44.7 (33.2/54.3) microg/l vs. 13.9 (10.4/23.9) microg/l; P = 0.01]). Therefore, the reduction of meningeal macrophages does not appear to attenuate inflammation in the subarachnoid space in experimental pneumococcal meningitis. Meningeal macrophages seem, however, to be important for the protection of neuronal tissue in bacterial meningitis.

IL-1beta immunoreactive neurons in the human hypothalamus: reduced numbers in multiple sclerosis.

Corticotropin-releasing hormone (CRH)-containing neurons in the paraventricular nucleus (PVN) in the hypothalamus of multiple sclerosis (MS) patients are hyperactivated. Since interleukin-1 (IL-1)beta is a powerful activator of CRH neurons, its immunohistochemical expression was studied in the postmortem hypothalamus of MS patients (n=11) and matched controls (n=11). Hypothalamic tissue of 10/11 MS patients showed demyelinating lesions that in many cases contained IL-1beta-immunoreactive (ir) macrophages and glial cells. In control subjects IL-1beta-ir was only sporadically found in glial cells. Interestingly, abundant IL-1beta-ir was also present in hypothalamic neurons. Neuronal IL-1beta co-localised with oxytocin and not with vasopressin or CRH. IL-1beta clearly yielded a less intense staining in neurons and numbers of IL-1-ir neurons in the PVN were 4.5-fold reduced in MS. We suggest that IL-1beta produced by activated glial cells in the hypothalamus of MS patients may contribute to the activation of the hypothalamic CRH neurons, while reduced expression of neuronal IL-1beta in MS patients may have consequences for neuroendocrine, behavioural or autonomic functioning.

Expression of interleukin-1 beta in rat dorsal root ganglia.

The expression of interleukin-1beta was examined in dorsal root ganglion (DRG) neurons from adult rats using non-radioactive in situ hybridization and immunocytochemistry. At all spinal levels, approximately 70% of the DRG neurons appeared to express IL-1beta mRNA; about 80% of these DRG neurons actually appeared to produce the IL-1beta protein at markedly varying levels. The expression of IL-1beta was found in large as well as in intermediate diameter sensory neurons but only sporadically in the population of small sensory neurons. The population of IL-1beta immunopositive sensory neurons included most of the large calretinin-positive Ia afferents, but only a few of the small substance P/CGRP positive sensory neurons. In situ hybridization staining for the detection of type 1 IL-1 receptor showed expression of this receptor by most of the sensory neurons as well as by supportive glial-like cells, presumably satellite cells. The functional significance of IL-1beta in the DRG neurons needs to be elucidated, but we speculate that IL-1beta produced by DRG neurons may be an auto/paracrine signalling molecule in sensory transmission.

Expression of the ED3 antigen on rat macrophages in relation to experimental autoimmune diseases.

Susceptibility to experimental autoimmune diseases (EAD) is rat strain dependent. Susceptible animals are reported to have a defective glucocorticoid response. Although many EAD are regarded as preferentially T cell-mediated, macrophages (M phi) play an important role in several different stages of these diseases. In this study we have investigated the possible effect of the disturbed hypothalamic-pituitary (HPA) axis on M phi phenotype. Therefore we studied M phi differentiation in several different rat strains, especially with regard to the M phi specific differentiation antigen as recognized by monoclonal antibody (mAb) ED3. This mAb is, in normal healthy rats, reactive with very restricted M phi subpopulations present in the lymphoid organs only. However, in autoimmune diseased tissues many of the infiltrated M phi are also ED3-positive. It appeared that M phi, in vitro derived from monocytes out of susceptible rat strains, showed a high ED3 expression in contrast to monocyte-derived M phi out of resistant rat strains. This difference in ED3 expression appeared to be T cell-mediated. Our results are suggestive for the fact that the impaired HPA-axis in EAD susceptible rat strains affects M phi differentiation. The relevance of the observed differences with respect to disease induction, maintenance, or suppression is discussed and obviously needs further investigation.

Liposome mediated affection of monocytes.

Dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, when injected intravenously, selectively eliminates all phagocytizing macrophages that are in direct contact with the blood circulation of the spleen and the liver. We examined whether Cl2MDP containing liposomes affect, in addition, rat monocytes and bone marrow macrophage precursors (BMMP's). In addition to Phosphatidylcholine-Cholesterol liposomes (PC liposomes), we also used mannosylated PC liposomes (PCMAN liposomes), which are reported to bind more efficiently to macrophages. Monocytes appeared to be affected 24 h after injections of 2 ml of Cl2MDP containing PC as well as PCMAN liposomes. In addition, almost no macrophages could be detected in one week cultures of blood leukocytes isolated from these animals; cultures from control animals contained +/- 50% macrophages. Bone marrow macrophage precursors did not appear to be affected.

Therapeutic effect of the D2-dopamine agonist bromocriptine on acute and relapsing experimental allergic encephalomyelitis.

We examined the effect of bromocriptine (BCR) treatment on the duration and severity of neurological symptoms of acute experimental allergic encephalomyelitis (EAE), an animal model for demyelinating diseases, particularly multiple sclerosis. To mimic the clinical situation, BCR treatment was started after the onset of clinical signs. Furthermore, the effect of BCR treatment on the course of a chronic relapsing form of EAE was studied. BCR was injected at daily intervals in a dose that resulted in sustained suppression of plasma concentrations of prolactin, a pituitary hormone that plays a role in immunoregulation. In acute EAE, BCR therapy reduced both severity and duration of the clinical signs. In chronic relapsing EAE, BCR treatment did not affect the severity and duration of the first attack, but reduced the duration of the subsequent, second attack. Thus, BCR treatment improves the clinical course in animals with ongoing disease. These findings may have implications for the search for new therapeutic approaches in multiple sclerosis.

Priming with interleukin-1beta suppresses experimental allergic encephalomyelitis in the Lewis rat.

Lewis rats exhibit multiple defects in their hypothalamus-pituitary-adrenal (HPA) system that are considered to play a causal role in the susceptibility of this strain to autoimmune diseases, i.e. experimental allergic encephalomyelitis (EAE). In the present study, we aimed to modulate the HPA response of the Lewis rat and establish its consequences for the susceptibility to EAE. Because in Wistar rats, single administration of interleukin (IL)-beta (priming) is known to induce long-lasting (weeks) sensitization of HPA responses to stressors and immune stimuli, Lewis rats were given a single dose of hIL-1beta or vehicle 1 week prior to induction of EAE by immunization with myelin basic protein (MBP). Subsequently, neurological deficits were monitored once daily. The results show that IL-1 priming markedly suppresses the neurological symptoms of EAE, without affecting the onset or duration of the disease. Measurement of vasopressin and corticotropin releasing hormone (CRH) in the external zone of the median eminence revealed that, as compared to Wistar rats, Lewis rats exhibit low vasopressin but identical CRH, and that IL-1 priming increases (0.001) vasopressin without affecting CRH stores, which is consistent with a shift to vasopressin-dominated control of adrenocorticotropic hormone (ACTH) secretion as described in Wistar rats under conditions of HPA hyper(re)activity. However, IL-1 priming did not affect a.m. corticosterone levels following immunization with MBP or during the clinical phase of EAE. IL-1 priming of Lewis rats attenuated the ACTH responses to an IL-1 challenge 11 days later, which may relate to an increase in resting corticosterone levels. Thus, the mechanisms underlying IL-1 induced suppression of EAE are not related to enhanced HPA responses. In addition, we did not find IL-1 priming-induced alterations in MBP-specific immunoglobulin (Ig)M, IgG1, IgGa and IgGb plasma titres, or gross alterations in T cell activation as reflected in spontaneous or concanavalin-induced T cell proliferation. We therefore speculate that IL-1-induced elevation of resting corticosterone levels may influence the development of EAE.

Substance P- and enkephalin-containing projections from the interpeduncular nucleus to the dorsal tegmental region in the rat.

A combination of fluorescent retrograde tracing and immunohistochemical staining for substance P (SP) and Leu-enkephalin (Enk) was used to study the projections from the interpeduncular nucleus (IP) to the dorsal tegmental region, i.e. the dorsal tegmental nucleus of Gudden, the dorsolateral tegmental nucleus and the caudal extension of the dorsal raphe nucleus. After injections of 'Granular Blue' (GB) in the dorsal tegmental region, followed one or two days later by a colchicine injection near the IP, and subsequently two days later by the immunohistochemical procedure, populations of neurons double labeled for Enk and GB, or SP and GB, cells that only showed GB labeling, and a number of cells stained only for one of the peptides could be identified in the rostral subnucleus of the IP. This study demonstrates the existence of both Enk- and SP-containing projections from the IP to the dorsal tegmental region.

Selective elimination of macrophages by dichlormethylene diphosphonate-containing liposomes suppresses experimental autoimmune neuritis.

The injection of liposome-encapsulated dichlormethylene diphosphonate (Cl2MDP) constitutes an effective method to selectively eliminate phagocytic cells from spleen, liver and the circulation. We evaluated the effect of Cl2MDP-liposomes on the course of actively induced and adoptively transferred experimental autoimmune neuritis (EAN), both animal models of the human Guillain-Barré syndrome. Injection of Cl2MDP-liposomes 11 and 13 days postimmunization (p.i.) of Lewis rats with bovine peripheral nerve myelin efficiently prevented clinical signs of EAN up to day 15 p.i., when all control animals were affected. Thereafter, EAN gradually also developed in Cl2MDP-liposome-treated rats, but until day 19 disease was significantly milder than in control rats injected with buffer-filled liposomes. Adoptive transfer EAN (AT-EAN) induced by injection of activated P2-specific T cells could be suppressed even more markedly by application of Cl2MDP-liposomes 1, 3, and 6 days after cell transfer. Efficient suppression of AT-EAN by Cl2MDP-liposomes rules out the possibility that EAN is prevented due to interference with the induction phase of this experimental disease and confirms that macrophages are important effector cells during EAN. Selective suppression of phagocytic cell function by drug-containing liposomes may hold promise as a novel treatment of demyelinating autoimmune diseases of the nervous system.

Macrophages in experimental autoimmune diseases in the rat: a review.

In the pathogenesis of most experimental autoimmune diseases T lymphocytes play a crucial role in the initiation, whereas macrophages are essential in the effector phase. This review deals with several methods to elucidate the exact role macrophages play in different stages of autoimmune models in the rat. By using monoclonal antibodies an inventory has been made on the different macrophage subsets that are present in the infiltrates of the affected tissues. That macrophages play a decisive role in provoking the clinical signs has been shown by several macrophage elimination studies. The severe tissue damage caused by macrophages is brought about by the release of inflammatory mediators. Especially interference with the production or action of these products could provide new therapeutical means.