Synergistic apoptosis induced by bacterial endotoxin lipopolysaccharide and high glucose in rat microglia - ScienceDirect
Microglia are the principal immune cells in the CNS and have critical role in host defense against invading microorganisms and neoplastic cells [10]. However, as with immune cells in other organs, microglia could also play a dual response, amplifying the effects of inflammation and mediating cellular degeneration [7]. Previous studies have indicated that diabetic neuropathy, including a decreased axonal transport, a reduced nerve conduction velocity, and an impaired axon regeneration, was observed in diabetic animals or patients [16]. These findings prompted us to investigate the possibility that increased levels of glucose could also affect other cell types within the CNS, such as microglia, and hence could contribute a role for the pathological consequences underlying diabetic neuropathy.
Lipopolysaccharide (LPS) that contributed to bacterial meningitis and septic shock, resulted in cell death or injury both in vivo and in vitro [5], [9], [11], [12]. Immune mediators, such as nitric oxide (NO) and the proinflammatory cytokines - tumor necrosis factor-α (TNF-α), induced by LPS were implicated in the mechanisms of LPS-induced cytotoxicity [5], [6]. Excessive formation of TNF-α as well as NO was also associated with lesioned white matter, microglial nodule formation, and vascular changes in the brain [3], [4], [10], [11], [12]. Thus, the major purpose of the study was to address the possibility of synergistic cytotoxicity of microglia that suffered from bacterial inflammatory insults in the presence of certain ambient conditions, such as increased levels of glucose.
Rat microglia-enriched cultures were prepared from whole brains of 1-day-old pups [18]. Microglia (1×107) were seeded in 75 cm2 culture flasks in 20 ml of a Dulbecco's modified Eagle's medium/F12 mixture (1:1) containing 10% heat-inactivated fetal bovine serum, 2 mM, l-glutamine; 1 mM, sodium pyruvate; 100 μM, non-essential amino acids; 50 units/ml, penicillin/streptomycin. Upon reaching confluence (day 19–20), microglia were shaken off and plated (105/well) into 24-well culture plates. Twenty-four hours later, cells were used for drug treatments. LPS (Escherichia coli 0111:B4), superoxide dimutase (SOD), and catalase (CAT) were purchased from Sigma Chemicals Company (St. Louis, MO, USA).
The production of NO was assessed as an accumulation of nitrite in the culture supernatants, using a colorimetric reaction with the Griess reagent [17]. Mitochondria function of microglia, in terms of, cell survival was determined by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cat. NO. M2128, Sigma). The concentration of TNF-α was measured with mouse TNF-α ELISA kit from Genzyme (Cambridge, MA, USA). DNA damage was detected by an in situ immunocytochemical assay, the terminal dUTP nick-end-labeling (TUNEL) technique, according to the manufacturer's recommendations (Roche Molecular Biochemicals, Cat. NO. 1684795, Indianapolis, IN, USA). The percentage of TUNEL-positive cells represents the ratio of TUNEL-positive cells and total cells from the same visual fields of the phase-contrast microscopy. The results are the mean ± standard error of mean of these experiments performed in duplicate. Statistical differences were first determined by one-way ANOVA followed by Bonferroni t-test for post-hoc multiple comparisons.
Fig. 1 (n=6) is a summary showing the effects resulted from a 24-h exposure of LPS and/or increased levels of D-glucose in primary cultured microglia. LPS (0.001 to 10 μg/ml) caused significant increases in both NO and TNF-α production, as compared to vehicle cultures. The MTT level was reduced only in response to 10 μg/ml LPS (Fig. 1A). Although the ambient levels of D-glucose concentrations were increased from 25 to 125 mM, there was no significant change in either NO or TNF-α production (Fig. 1B). However, the decreased MTT levels following D-glucose addition were in a concentration-dependent manner (Fig. 1B). It was unlikely that the effects following D-glucose addition was mainly due to the changes in osmolarity rather than glucose itself since sucrose (125 mM) was used as a vehicle control. There was no significant difference between the control and sucrose cultures. To determine whether a synergistic cytotoxicity resulted from LPS in the presence of increased levels of D-glucose, cells were pretreated with D-glucose for 1 h and LPS for a following 24 h. As shown in Fig. 1C (n=6), D-glucose (125 mM) alone and LPS (1 μg/ml) alone only decreased MTT levels down to 64.3±5.5% and 82.9±6.6%, respectively. When microglia were exposed to a combined treatment of D-glucose (125 mM) with LPS (1 μg/ml), the MTT levels were synergistically reduced down to 32.4±7.2%.
Fig. 1. The changes of NO production, TNF-α release, and cell viability in primary microglia cultures. (A) 1, 2, 3, 4, 5 and 6 represent vehicle, 0.001, 0.01, 0.1, 1, and 10 μg/ml LPS, respectively. *P<0.05 compared to the cultures treated with vehicle cultures. (B) 1, vehicle; 2, 125 mM sucrose; 3, 4, 5, 6 and 7 represent 25, 50, 75, 100, and 125 mM D-glucose, respectively. *P<0.05 compared to the cultures treated with vehicle cultures. (C) 1, vehicle; 2, 1 μg/ml LPS; 3, 125 mM D-glucose; 4, 1 μg/ml LPS+25 mM D-glucose; 5, 1 μg/ml LPS+50 mM D-glucose; 6, 1 μg/ml LPS+75 mM D-glucose; 7, 1 μg/ml LPS+100 mM D-glucose; 8, 1 μg/ml LPS+125 mM D-glucose. *P<0.05 compared to the cultures treated with 1 μg/ml LPS.
To determine the mode of this synergistic cytotoxicity in microglia, phase-contrast microscopy and nuclear chromatin staining with TUNEL techniques were used. Phase-contrast analysis of microglia exposed to LPS (1 μg/ml) in the presence of D-glucose (125 mM) showed microglia with shrunken and irregularly shaped cell bodies (data not shown). TUNEL staining further indicated fragmentation and condensation of chromatin in microglia treated with LPS (1 μg/ml) plus D-glucose (125 mM) (Fig. 2). In addition, the number of apoptotic microglia (43.8±3.2%, n=6) following LPS (1 μg/ml) plus D-glucose (125 mM) was significantly different to the experiments subjected with LPS (1 μg/ml) alone (31.7±2.9%, n=6) or D-glucose (125 mM) alone (27.5±2.2%, n=6).
Fig. 2. Morphological in situ assay of DNA damage in primary microglia cultures. TUNEL-staining micrographs of microglial cells (A–D) were taken from the same experiment 24 h after exposure to the following treatments: (A), vehicle; (B), D-glucose (125 mM); (C), LPS (1 μg/ml); (D), D-glucose (125 mM)+LPS (1 μg/ml).
To investigate whether oxidative free radicals played a role in this synergistic cytotoxicity, traditional free radical scavengers, SOD / CAT were used. As indicated in Fig. 3 (n=6), the use of SOD (100 units/ml) / CAT (50 units/ml) significantly attenuated the magnitudes of synergistic cytotoxicity, but not the production of NO or TNF-α.
Fig. 3. Attenuation of the magnitudes of the synergistic cytotoxicity by free radical scavengers. 1, vehicle; 2, SOD/CAT; 3, 1 μg/ml LPS+125 mM D-glucose; 4, SOD/CAT+1 μg/ml LPS+125 mM D-glucose. *P<0.05 compared to the cultures treated with 1 μg/ml LPS+125 mM D-glucose.
Glucose is the major energetic substrate for immunocomponent cells like macrophages, monocytes, and microglia. Glucose uptake was tremendously increased in activated immunocomponent cells during LPS exposure [13], [14]. Thus, an increased utilization of glucose in microglia during LPS exposure facilitated the metabolic turnover rate of mitochondria. This enhanced biochemical cascade of events in mitochondria led to an increase of toxic factor production (e.g. oxidative free radicals), and hence eventually made microglia themselves more susceptible to surrounding insults (e.g. LPS) [1].
Activated microglia during bacterial inflammation within the CNS promoted the generation of reactive oxygen intermediates [4], [9]. Microglia have been suggested to be the first line of defensive cells in the CNS [10]. The activation of microglia occurred in nearly every type of neurological disorders and was often associated with an increased production of various cytokines (e.g. TNF-α) and reactive oxygen species (e.g. NO) [2], [3], [4], [8], [9], [15]. However, it was unlikely that TNF-α and NO played a major role for this synergistic cytotoxicity seen in the present study.
In summary, the present results suggest that increased ambient levels of glucose rendered microglia vulnerable to surrounding LPS insults and this synergistic apoptosis was, at least in part, mediated by oxidative free radicals. The findings here may be important in certain patho-physiological implications in which hyperglycemia is deleterious to the physiological functions contributed by microglia, and may provide new insight into a novel therapeutic intervention.
Acknowledgements
This study was supported in part by National Health Research Institute, Taiwan, ROC; Grant NHRI-EX90–8909BP.