Talma Brenner

Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppression of Th1 and Th17 Responses

The α7 nicotinic acetylcholine receptor (nAChR) was recently described as an anti-inflammatory target in both macrophages and T cells. Its expression by immune cells may explain the epidemiological data claiming a negative link between cigarette smoking and several inflammatory diseases. In this study, we determined the immunological effects of α7 nAChR activation by nicotine. Our results indicate that the α7 nAChR is expressed on the surface of CD4+ T cells and that this expression is up-regulated upon immune activation. Nicotine reduced T cell proliferation in response to an encephalitogenic Ag, as well as the production of Th1 (TNF-α and IFN-γ) and Th17 cytokines (IL-17, IL-17F, IL-21, and IL-22). IL-4 production was increased in the same setting. Attenuation of the Th1 and Th17 lineages was accompanied by reduced T-bet (50%) and increased GATA-3 (350%) expression. Overall, nicotine induced a shift to the Th2 lineage. However, α7−/−-derived T cells were unaffected by nicotine. Furthermore, nicotine reduced NF-κB-mediated transcription as measured by IL-2 and IκB transcription. In vivo, administration of nicotine (2 mg/kg s.c.) suppressed the severity of CD4+ T cell-mediated disease experimental autoimmune encephalomyelitis. α7−/− mice were refractory to nicotine treatment, although disease severity in those animals was reduced, due to impairment in Ag presentation. Accordingly, CD4+ and CD11b+ cells infiltration into the CNS, demyelination, and axonal loss were reduced. Our data implicate a role for the α7 nAChR in immune modulation and suggest that α7 nAChR agonists may be effective in the treatment of inflammatory disorders.

Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors

We investigated the anti-inflammatory effects of acetylcholinesterase inhibitors (AChEI) at the cellular and molecular levels. AChEI suppressed lymphocyte proliferation and pro-inflammatory cytokine production, as well as extracellular esterase activity. Anti-inflammatory activity was mediated by the alpha7 nicotinic acetylcholine receptor (neuronal); the muscarinic receptor had the opposite effect. Treatment of the central nervous system (CNS) inflammatory disease, experimental autoimmune encephalomyelitis (EAE), with EN101, an anti-sense oligodeoxynucleotide, targeted to AChE mRNA, reduced the clinical severity of the disease and CNS inflammation intensity. The results of our experiments suggest that AChEI increase the concentration of extracellular acetylcholine (ACh), rendering it available for interaction with a nicotinic receptor expressed on lymphocytes. Our findings point to a novel role for AChEI which may be relevant in CNS inflammatory diseases such as EAE and multiple sclerosis. They also emphasize the importance of cholinergic balance in neurological disorders, such as Alzheimers disease and myasthenia gravis, in which these drugs are used.

Humoral and cellular immune responses to Copolymer 1 in multiple sclerosis patients treated with Copaxone

Humoral and cellular immune responses were followed in multiple sclerosis patients treated with Copolymer 1 (Cop1, glatiramer acetate, Copaxone) who participated in three different clinical trials. All patients (130) developed Cop1 reactive antibodies, which peaked at 3 months after initiation of treatment, decreasing at 6 months and remaining low. IgG1 antibody levels were 2-3-fold higher than those of IgG2. The proliferative response of Peripheral Blood Mononuclear Cells (PBMC) to Cop1 was initially high and gradually decreased during treatment. Antibodies and T cell responses to MBP were low and did not change significantly during the treatment. The humoral and cellular immunological responses to Cop1 do not correlate with the side effects and do not affect its therapeutic activity. The preferential production of IgG1 over IgG2 antibodies may indicate that Th2 responses are involved in mediating the clinical effect of Cop1.

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare “readthrough” acetylcholinesterase variant AChE‐R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE‐R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE‐R in blood and muscle yet leaves unaffected the synaptic variant AChE‐S. Whereas animals treated with placebo or conventional anticho‐linesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA‐targeted therapeutics for chronic cholinergic malfunctioning.—Brenner, T., Hamra‐Amitay, Y., Evron, T., Boneva, N., Seidman, S., Soreq, H. The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis. FASEB J. 17, 214–222 (2003)

Suppression of experimetal autoimmune encephalomyelitis by cannabinoids

The effect of delta 8-THC on experimental autoimmune encephalomyelitis (EAE) was examined. delta 8-THC is an analogue of delta 9-THC, the psychoactive component of marijuana. It is more stable and less psychotropic than delta 9-THC and like the latter it binds to the brain cannabinoid receptor. Two strains of rats were inoculated for EAE, and delta 8-THC (40 mg/kg) was administered for up to 21 days. delta 8-THC significantly reduced the incidence and severity of neurological deficit in both rat strains. The beneficial influence of delta 8-THC only occurred on oral administration and not with parenteral injection. Serum corticosterone levels were twofold elevated in rats with EAE chronically treated with delta 8-THC. These results suggest that suppression of EAE by cannabinoids may be related to their effect on corticosterone secretion.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

Mast cells in experimental allergic encephalomyelitis: characterization, distribution in the CNS and in vitro activation by myelin basic protein and neuropeptides.

Mast cells (MC) have been implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE). In order to further evaluate their role, several morphological and functional studies were performed. Semiquantitative counts of histological sections showed a significant reduction in MC numbers in EAE brains. In addition, a higher proportion of EAE MC (about 50-70%) appeared degranulated compared with about 20% degranulation in controls. Central nervous system (CNS) MC exhibited staining properties of connective tissue MC and about 98% of them, both in diseased and control rats, were located in the thalamus. They were not present in the spinal cord and did not relate to EAE lesions. In vitro incubation of peritoneal MC (of connective tissue phenotype) with either MBP, or with neuropeptides such as substance P or bradykinin resulted in release of beta-hexosaminidase and histamine. The latter responses were similar in both EAE and control rats. It is suggested that the decrease in number and in granular content of CNS MC in EAE may reflect prior in vivo activation. The fact that MC were activated by MBP and by neuropeptides in vitro suggests a possible mechanism of MC activation in EAE.

Suppression of neuroinflammation and immunomodulation by the acetylcholinesterase inhibitor rivastigmine

In this study we determined the influence of cholinergic up-regulation by rivastigmine, an acetylcholinesterase inhibitor, on central nervous system inflammation. Neuroinflammation was induced in experimental autoimmune encephalomyelitis (EAE). Rivastigmine markedly ameliorated clinical symptoms of EAE and the spatial memory deficits induced by EAE. It also reduced demyelination, microglia activation and axonal damage. Rivastigmine decreased the reactivity of encephalitogenic T-cells and the production of pro-inflammatory cytokines (TNF-alpha, IFN-gamma and IL-17) without affecting IL-10 production. These effects were abolished by alpha7 nicotinic acetylcholine receptor antagonists. Antigen presentation was also affected by this treatment. Thus, rivastigmine treatment had immunomodulatory activity in EAE.

Differential effects of central and peripheral nerves on macrophages and microglia

The poor ability of injured central nervous system (CNS) axons to regenerate has been correlated, at least partially, with a limited and suppressed postinjury inflammatory response. A key cell type in the inflammatory process is the macrophage, which can respond in various ways, depending on the conditions of stimulation. The aim of this study is to compare the activities of macrophages or microglia when encountering CNS and peripheral nervous systems (PNS), on the assumption that nerve‐related differences in the inflammatory response may have implications for tissue repair and thus for nerve regeneration. Phagocytic activity of macrophages or of isolated brain‐derived microglia was enhanced upon their exposure to sciatic (PNS) nerve segments, but inhibited by exposure to optic (CNS) nerve segments. Similarly, nitric oxide production by macrophages or microglia was induced by sciatic nerve segments but not by optic nerve segments. The previously demonstrated presence of a resident inhibitory activity in CNS nerve, could account, at least in part, for the inhibited phagocytic activity of blood‐borne macrophages in CNS nerve as well as of microglia resident in the brain. It seems that the CNS microglia are reversibly immunosuppressed by the CNS environment, at least with respect to the activities examined here. It also appears from this study that the weak induction of early healing‐related activities of macrophages/microglia in the environment of CNS might explain the subsequent failure of this environment to acquire growth‐supportive properties in temporal and spatial synchrony with the needs of regrowing axons. GLIA 23:181–190, 1998.

Stimulation of tumor necrosis factor-α production by mycoplasmas and inhibition by dexamethasone in cultured astrocytes

Elevated levels of tumor necrosis factor-alpha (TNF alpha) and other cytokines and eicosanoids in the central nervous system (CNS) have been noted in several human neurologic diseases, including multiple sclerosis and AIDS dementia. Recently it was shown that glial cells, especially astrocytes, are a major source of cytokines and eicosanoids. In the present study we have shown that astrocytes derived from fetal rat brain triggered by mycoplasmas produce TNF alpha and prostaglandin E2 (PGE2). Addition of mycoplasma (Mycoplasma capricolum isolated from sheep and M. fermentans KL-4 from human) at a concentration of 1-50 micrograms protein/ml (2 x 10(7)-10(9) colony forming units/ml), as well as lipopolysaccharide (5 micrograms/ml), led to a 200-500-fold increase in TNF alpha and a 2.5-4.5-fold increase in PGE2 production. Preincubation of the cells with the synthetic glucocorticoid, dexamethasone (2 x 10(-5)-2 x 10(-8) M), as well as with the natural hormone, corticosterone, markedly inhibited the secretion of both TNF alpha and PGE2. Thus, mycoplasmas can be added to the wide variety of agents that stimulate glial cells to produce cytokines and eicosanoids, and may contribute to various CNS pathological manifestations. In addition, the ability of glucocorticoids to inhibit particularly the stimulated productions of TNF alpha and PGE2 may explain at least in part the therapeutic benefit of these agents in CNS inflammation and demyelination.