Microglia suppress the secondary progression of autoimmune encephalomyelitis.

Secondary progressive multiple sclerosis (SPMS) is an autoimmune disease of the central nervous system (CNS) characterized by progressive motor dysfunction, sensory deficits, and visual problems. The pathological mechanism of SPMS remains poorly understood. In this study, we investigated the role of microglia, immune cells in the CNS, in a secondary progressive form of experimental autoimmune encephalomyelitis (EAE), the mouse model of SPMS. We induced EAE in nonobese diabetic mice and treated the EAE mice with PLX3397, an antagonist of colony stimulating factor-1 receptor, during secondary progression in order to deplete microglia. The results showed that PLX3397 treatment significantly exacerbated secondary progression of EAE and increased mortality rates. Additionally, histological analysis showed that PLX3397 treatment significantly promoted inflammation, demyelination, and axonal degeneration. Moreover, the number of CD4+ T cells in the spinal cord of EAE mice was expanded due to PLX3397-mediated proliferation. These results suggest that microglia suppressed secondary progression of EAE by inhibiting the proliferation of CD4+ T cells in the CNS.

Zinc-related actions of sublethal levels of benzalkonium chloride: Potentiation of benzalkonium cytotoxicity by zinc.

Benzalkonium chloride (BZK) is a common preservative used in pharmaceutical and personal care products. ZnCl2 was recently reported to significantly potentiate the cytotoxicity of some biocidal compounds. In the present study, therefore, we compared the cytotoxic potency of BZK and then further studied the Zn2+-related actions of the most cytotoxic agent among BZK, using flow cytometric techniques with appropriate fluorescent probes in rat thymocytes. Cytotoxicity of benzylcetyldimethylammonium (BZK-C16) was more potent that those of benzyldodecyldimethylammonium and benzyldimethyltetradecylammonium. ZnCl2 (1-10 µM) significantly potentiated the cytotoxicity of BZK-C16 at a sublethal concentration (1 µM). The co-treatment of cells with 3 µM ZnCl2 and 1 µM BZK-C16 increased the population of both living cells with phosphatidylserine exposed on membrane surfaces and dead cells. BZK-C16 at 0.3-1.0 µM elevated intracellular Zn2+ levels by increasing Zn2+ influx, and augmented the cytotoxicity of 100 µM H2O2. Zn2+ is concluded to facilitate the toxicity of BZK. We suggest that the toxicity of BZK is determined after taking extracellular (plasma) and/or environmental Zn2+ levels into account.

Chlorhexidine possesses unique cytotoxic actions in rat thymic lymphocytes: Its relation with electrochemical property of membranes.

Chlorhexidine (CHX) is an antibacterial agent used in various types of pharmaceutical products. Therefore, CHX is easily found around us. Owing to its positive charge, the electrochemical property of cell membranes was assumed to be a key point of cytotoxic action of CHX. Depolarization of membranes attenuated the cytotoxic action of CHX in rat thymic lymphocytes. CHX interfered with annexin V binding to membranes. Manipulations to induce exposure of phosphatidylserine on the outer membrane surface augmented the cytotoxic action of CHX, indicating that changes in the electrochemical property of membranes affected the cytotoxic action of CHX. Hence, CHX might kill cells physiologically undergoing apoptosis, resulting instead in necrotic cell death. However, the threshold CHX concentration in this in vitro study was slightly higher than blood CHX concentrations observed clinically. Therefore, these results may support the safety of CHX use although CHX possesses unique cytotoxic actions described in this study.

Zinc increases vulnerability of rat thymic lymphocytes to arachidonic acid under in vitro conditions.

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca(2+). In the present study, the Zn(2+)-related cytotoxicity of ARA was studied by a flow cytometric technique with appropriate fluorescent probes in rat thymocytes. Addition of 10 µM ZnCl2 enhanced the increase in cell lethality induced by 10 µM ARA. The removal of Zn(2+) by Zn(2+) chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn(2+) is suggested to be involved in ARA cytotoxicity. ARA at 3-10 µM elevated intracellular Zn(2+) level. The Zn(2+) chelators attenuated the ARA-induced increase in intracellular Zn(2+) level while ARA significantly increased intracellular Zn(2+) level in the presence of 3 µM ZnCl2, suggesting the involvement of external Zn(2+). Zn(2+) reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn(2+) levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful.

Increase in intracellular Ca(2+) level by phenylsulfamide fungicides, tolylfluanid and dichlofluanid, in rat thymic lymphocytes.

Tolylfluanid, a phenylsulfamide fungicide, is one of the many pesticides that are frequently detected in crops. Therefore, its health risk is a concern. Micromolar concentrations of tolylfluanid induce chromosomal aberrations and micronuclei in mammalian lymphocytes. The findings prompted us to study the cellular actions of tolylfluanid and another frequently detected pesticide, dichlofluanid, at submicromolar and micromolar concentrations. Of the cellular actions of chemicals, the action on cellular Ca(2+) homeostasis is important since Ca(2+) is involved in cell signaling and death. Consequently, in this study, the effects of phenylsulfamide fungicides were examined on rat thymocytes by using fluorescent probes in order to further characterize the cellular actions of phenylsulfamide fungicides. Both phenylsulfamide fungicides exhibited biphasic, early and late, increase in intracellular Ca(2+) levels. The early phase was dependent on intracellular Ca(2+) release and increased membrane Ca(2+) permeability. The late phase was owing to Ca(2+) influx via activation of store-operated Ca(2+) channels and the further increase of membrane ionic permeability. Voltage-dependent Ca(2+) channels were not involved. The increases in intracellular Ca(2+) levels by phenylsulfamide fungicides were observed at drug concentrations of 0.1 µM or more (up to 10 µM). Thus, it is plausible that micromolar concentrations of phenylsulfamide fungicides deregulate intracellular Ca(2+) homeostasis in rat thymocytes. Both phenylsulfamide fungicides at 10 µM promoted the transition from intact living cells to living cells with phosphatidylserine-exposed membranes. This was not the case for phenylsulfamide fungicides at 3 µM. The potency of tolylfluanid was similar to that of dichlofluanid. Although the information on residual concentrations of tolylfluanid and dichlofluanid is very limited, their residual concentrations do not reach micromolar levels. It is unlikely that humans will develop adverse effects on exposure to phenylsulfamide fungicides under present environmental conditions.

Zn(2+)-dependence of the synergistic increase in rat thymocyte cell lethality caused by simultaneous application of 4,5-dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) and H2O2.

4,5-Dichloro-2-octyl-4-isothiazolin-3-one (DCOIT) is an antifouling agent that is an alternative to organotins such as tributyltin (TBT). Because DCOIT decreases catalase activity, it may increase the susceptibility of cells to oxidative stress. We examined the effects of DCOIT on rat thymocytes suffering from oxidative stress induced by H2O2. The simultaneous application of DCOIT and H2O2 induced a synergistic increase in cell lethality that was completely suppressed by chelating intracellular Zn(2+). Intracellular Zn(2+) concentration was increased by DCOIT at concentrations ranging from 0.1 µM to 3 µM. Although the increase in cell lethality produced by DCOIT alone was less than that produced by TBT alone, a synergistic increase was not induced by the combination of TBT and H2O2. Therefore, these results suggest that DCOIT increases vulnerability to oxidative stress and is more cytotoxic than TBT when oxidative stress is induced by H2O2.