Protective Mechanism of Melatonin on Kainic Acid Induced Immune Modulatory Effect on Lymphocytes Derived from Mouse Spleen

Toxicity occurs when cells develop inflammation due to exposure to several toxic substances. The study was aimed to assess the damage (toxicity) induced by kainic acid on splenic lymphocyte and enriched T lymphocyte under in vitro conditions and the protective role of exogenous melatonin against this damage (toxicity). The study included assessment of inflammatory mechanism by the expression of immune modulatory cytokine mediators using real time PCR. Oxidative stress (reactive oxygen species) and nitrosative stress (reactive nitrogen species) were also studied to determine the free radical production. Interestingly, kainic acid caused severe splenic lymphocytes and enriched T lymphocyte damage which was evident from deleterious alterations in various parameters. Kainic acid treatment (1 mM) resulted in increased mRNA expression of cytokines like tumour necrosis factor-beta, interleukin 6, interleukin 1, interferon gamma, mitogen-activated protein kinase gene-14, inducible nitric oxide synthase and decreased interleukin 10 mRNA expression. Tritiated (3H) Thymidine Incorporation study signifies that kainic acid treatment (1 mM) increased the proliferation of splenic and enriched T lymphocytes. These changes were normalized by exogenous administration of melatonin (0.25-1.0 mM) in combination with kainic acid. Flow Cytometry Analysis using Annexin V apoptosis assay kit revealed an increase in apoptotic and necrosis (double positive cells) in splenic lymphocytes treated with kainic acid alone. However, melatonin treated in combination with kainic acid, showed attenuation to apoptosis and necrosis on splenic lymphocytes. This study depicts that kainic acid induced inflammatory toxicity could be attenuated by exogenous melatonin treatment as evident by the decreased levels of the inflammatory cytokines, immune reactions and free radical production. Protective Mechanism of Melatonin on Kainic Acid Induced Immune Modulatory Effect on Lymphocytes Derived from Mouse Spleen


Introduction
Kainic acid (KA) is an agonist for a subtype of ionotropic glutamate receptor, and administration of KA has been shown to increase production of reactive oxygen species, mitochondrial dysfunction, and apoptosis in neurons in many regions of the brain, particularly in the hippocampal sub regions of CA1 and CA3, and in the hilus of dentate gyrus (DG) [1]. This is associated with intense stimulation of microglia in the brain, monocyte infiltration, oxidative stress, induction of expression of genes such as IL-1β, TNF-α, iNOS and COX-2 and intense inflammatory responses [2][3][4]. High levels of free radicals are known to damage cells [5]. KA influences immune system, generates excess free radicals and reduces the levels of anti-oxidant enzymes and induces oxidative stress not only in the brain but also in the liver and kidneys of mice [6].
Melatonin is a free radical scavenger, anti-oxidant and immune modulator [7][8][9]. It is known to protect cells from mitochondrial dysfunction and related disorders caused by oxidative stress [10]. It is also known to ameliorate KA-induced oxidative stress, neuro-toxicity, DNA damage etc. KA causes an increase in the levels of 8-hydroxydeoxyguanosine in brain and liver; melatonin reduces this increase [11].
Lesions induced by KA on cerebellar fastigial nucleus participates in the modulation of lymphocyte functions and that the hypothalamus and sympathetic nerves innervating lymphoid organs are involved in this neuro immunomodulation [12]. KA-induced hippocampal neurodegeneration influences adaptive immune response and B cell and T cell subsets may contribute differently to the pathogenesis [13].
IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation [14].
Fundamental inflammatory responses such as the induction of cyclooxygenase type 2, increases expression of adhesion molecules, or synthesis of nitric oxide are indistinguishable responses of both IL-1 and TLR ligands. Both families nonspecifically affect antigen recognition and lymphocyte function. IL-1 beta is the most studied member of the IL-1 family because of its role in mediating autoinflammatory diseases [15]. IL-6 exerts stimulatory effects on T-and B-cells, thus favoring cytotoxic agent and modulates immune responses and inflammation p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is activated by chemical and physical stress and regulates immune responses [18]. Studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli [19].
The rational of this study was to elucidate the effect and mechanism of action of KA induced inflammatory effect on splenic lymphocytes and enriched T lymphocytes, ensuing attenuation by the treatment of melatonin.
The effect of KA on mRNA expressions of cytokines IL-10, IL-6, TNF-β and INF γ on splenic lymphocytes and enriched T lymphocytes isolated from spleen is not well established. Therefore, influence of KA on these cytokines, signaling pathways involved during inflammation and the role of melatonin on the same was also studied.

Animals
Swiss albino mice were selected for this study. The animals were procured from the Division of Laboratory Animal Sciences of Biomedical Technology Wing (BMT), Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Thiruvananthapuram, Kerala, India with prior approval from Institutional Animal Ethics Committee and in accordance with approved Institutional protocol. Animals were maintained in a 12 h light and dark cycle at controlled environmental conditions of temperature (22 ± 3°C) and humidity (50-70%). They were fed with standard pellet diet and provided water ad libitum.
All animals were handled with care, without causing pain or distress. The care and management of the animals comply with the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India.

Collection of splenic lymphocytes and enriched T lymphocytes
Six healthy swiss albino mice were sacrificed by cervical dislocation. Spleen was dissected out immediately and washed with sterile Phosphate Buffered Saline (PBS). The splenic lymphocytes were collected by disrupting the isolated spleen in PBS as described elsewhere [20]. The enriched T lymphocytes were isolated from the single cell suspension of the splenic lymphocytes, isolated as above in the automated cell separator (ROBOSEP) using the kit according to the manufacture's protocol.
Similar experiment was carried out independently with enriched T lymphocytes isolated from the single cell suspension of splenic lymphocytes as mentioned above. All the above groups were used to determine various biochemical and molecular parameters.

Tritiated (3H) thymidine incorporation assay using splenic lymphocytes and enriched T lymphocytes
Isolated spleen from mice (as mentioned above) was used for the isolation of splenic lymphocytes and enriched T lymphocytes to study the cell proliferation by tritiated thymidine incorporation assay in cell culture. Viability of splenic lymphocytes and enriched T lymphocytes were assessed using trypan blue dye exclusion method [21]. Splenic lymphocytes and enriched T lymphocytes were cultured at a density of 2×10 5 and 1×10 4 cells/ml respectively in a 96 well plate with RPMI-1640 medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 Units/ml) for 24 h at 37°C in a CO 2 incubator. After 24 h of incubation the cultured wells were treated with1 mg/ml physiological saline (normal control), kainic acid alone (KA-1 mM), melatonin alone (MLT-1 mM), KA and MLT in combinations in various concentrations (as mentioned in experimental design) were added in the respective 96 well plate. After 48 h of incubation at 37°C, the culture treated wells were labeled with 3H-thymidine at a concentration of 1 μci/ml and incubated further for 24 h at 37°C. Cells were harvested after 72 h and radioactivity in terms of counts per minute (cpm) were measured by Liquid Scintillation Counter (Triathler, Hidex).

Real time PCR analysis for determining m-RNA expression of cytokines, iNOS and MAPK 14 gene on splenic lymphocytes and enriched T lymphocytes
Single cell splenic lymphocytes suspension and isolated enriched T lymphocytes were cultured separately at a density of 2×10 5 and 1×10 4 cells/ml respectively in a 35 cm 2 culture dish with RPMI-1640 medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 Units/ml) for 24 h at 37°C in a CO 2 incubator for the isolation of total RNA. After 24 h of incubation, various concentrations of KA and melatonin in combinations (as mentioned in Experimental Design) were added in the respective culture dish. Cells treated with 1 mg/ml physiological saline served as normal control. After 48 h of incubation, total RNA was isolated from cultured splenic lymphocytes and enriched T lymphocytes using RNeasy lipid tissue mini kit (Qiagen, Germany). Following the manufacture's protocol quantity and purity was analyzed spectrophotometrically. 100 ng of mRNA were used for cDNA synthesis of IL-6, IL-10, IL-1, TNF-β, MAPK 14, iNOS, and GAPDH in a reaction volume of 20 μl using RT2 first strand kit (Qiagen, Germany). The synthesis was carried out in Eppendrof master cycler, Germany.
The mouse oligo nucleotide forward and reverse primer sequence used to determine specific mRNA gene expressions were procured from Eurogenetc, Belgium are depicted in Table 1. The real time PCR reaction was carried out with RT2 SYBR Green ROX qPCR master mix (Qiagen, Germany) of total reaction volume of 25 ml; real time PCR amplifications were done using a Chromo 4 System, Bio-Rad (MJ Research, CA) for 40 cycles as per manufacture's protocol. Glyceraldehyde Phosphate Dehydrogenase (GAPDH) was used as the house keeping gene. The level of gene expression is reported as the ratio between the mRNA level of the target gene and the GAPDH, a reference gene using the comparative 2δδct method [22].

Estimation of oxidative stress by reactive oxygen species (ROS) generation by DCFH-DA (2'7'-dicholoroflorescien acetate) on splenic lymphocytes and enriched T lymphocytes
ROS generation in splenic lymphocytes and enriched T lymphocytes treated with KA, MLT and various combinations of KA and MLT combinations as mentioned in the experimental design were assayed using DCFH-DA (2'7'-dicholoroflorescien acetate), a non fluorescent, cell permeant dye. DCFH-DA is converted to fluorescent DCF by the free radicals generated by the cell during oxidative stress. This can be monitored by using fluorescent spectrophotometer. The isolated cells were treated with DCFH-DA according to the protocol of Benedetti et al. [23]. Splenic lymphocytes and enriched T lymphocytes were incubated with DCFH-DA for 30 minutes independently. The supernatant was discarded and different concentrations of KA and MLT (as mentioned in experimental design) were added and incubated for 4 h. After the period, fluorescence was read with excitation wavelength at 450 nm and emission wavelength at 535 nm.
KA alone and MLT alone treated controls were also used for the study. Untreated cells served as normal control.

Nitrite determination to assess nitrosative stress by reactive nitrogen species (RNS) generation using Griess reagent on splenic lymphocytes and enriched T lymphocytes
Production of nitric oxide free radical (NO) by inducible nitric oxide synthase (iNOS) was analyzed. Auto-oxidation of NO • results in production of nitrite which is measured by using Griess reagent [24]. Splenic lymphocytes and enriched T lymphocytes were treated with KA, MLT and various concentrations of KA and MLT in combinations as mentioned in the experimental design. Treated culture was incubated for 24 h independently. Added equal volume of Griess reagent (Sigma, USA) to the cultured cells and further incubated at room temperature for 10 minutes. Standard curve was plotted using sodium nitrite at different concentrations. Absorbance was measured at 540 nm. The concentration of nitrite in the sample was determined by the standard curve.

Live/dead/apoptotic assay using isolated splenic lymphocytes by flow cytometry ananlysis on splenic lymphocytes
Apoptosis assay was carried out to evaluate the cytotoxicity and apoptosis induced by kainic acid and the attenuation of apoptosis induced by the treatment of melatonin in various concentrations on splenic lymphocytes using Vybrant Apoptosis Assay Kit (Molecular Probes). After 72 h, culture of splenic lymphocytes and enriched T lymphocytes, cells were harvested (as described earlier) and washed with PBS later stained with Annexin V-Alexa flour 488 and Propidium iodide (PI) according to manufacturer's protocol. Analysis was carried out by Flow Cytometry and percentages of live, apoptotic and dead cells (necrotic) were estimated using BD FACS Diva software.

Statistical analysis
All the samples were run in triplicates; statistical comparison was done with three or more groups using one-way Analysis of Variance (ANOVA) followed by Dunnetts' test. P values<0.005 and <0.05 were considered significant. Kainic acid control group was compared with normal control group. Kainic acid and melatonin treated combination groups were compared with the Kainic acid control group to assess the statistical significance using ANOVA.

Effect of melatonin on kainic acid induced changes in mRNA expressions of iNOS and MAPK 14 gene on splenic lymphocytes and enriched T lymphocytes from mouse spleen using real time PCR
As shown in Figure

Counter effect of melatonin on ROS and RNS production induced by kainic acid on splenic lymphocytes and enriched T lymphocytes isolated from mouse spleen
KA (1 mM) treatment increased oxygen free radicals significantly in both splenic lymphocytes and enriched T lymphocytes. MLT in a concentration dependent manner attenuated this increase in reactive

Discussion
Kainic acid is a neurotoxin isolated from sea weed. When injected into animals it mimics the action of the excitatory amino acid neurotransmitter, glutamate [25]. Kainic acid produces acute and subacute epileptiform activity that can last for days. Ultimately, it causes widespread irreversible neuropathological changes that involve both neurons and glia [26]. Kainic acid binds to and activates a subtype of ionotropic glutamate receptor. In addition to inducing brain lesions directly, probably via processes involving free radicals, kainic acid also provokes the discharge of potentially neurotoxic quantities of glutamate from nerve endings [27].
In earlier studies the authors have demonstrated KA induced deleterious effects on the antioxidant enzymes as well as on antioxidant parameters both in vitro and in vivo on brain tissue. Such an effect was abolished with MLT treatment in a concentration dependent manner. The study also showed KA induced alterations in cytokine mRNA expression as well as nucleotide alterations were observed in the cytochrome b sequence of mouse mtDNA. These effects were attenuated by MLT treatment in a concentration dependent manner [20]. The immune modulatory effect of KA and MLT on lymphocytes is not well documented yet. In the present study authors provide valuable information on the effect of KA on lymphocytes.
Given that free radicals are likely involved in the neurodestructive processes of kainic acid, soon after melatonin was discovered as a free radical scavenger, it was tested for its antiexcitatory efficacy [28].
The authors report here that KA had drastic deleterious effect on lymphocytes which was attenuated by the treatment of MLT. In this study the authors documented that melatonin did not directly inhibit the action of kainic acid on lymphoctes but had an anti-inflammatory and antioxidant mechanism which was evident by the expression mRNA of MAPK 14, iNOS and other inflammatory cytokine levels, However attenuation of free radical species and proinflammatory cytokines was resulted by MLT treatment in a concentration dependent manner. In vivo as well, melatonin reduced the ability of kainic acid to kill neurons in both the cerebellum and in the hippocampus [29,30]. Moreover, melatonin reduced the associated neural lipid peroxidation as well as preventing the death of the majority of the rats that received kainic acid [31]. The authors of these reports were convinced that the beneficial effects of melatonin against neurotoxicity of kainic acid were due to melatonin's ability to readily enter the brain and scavenge free radicals and stimulate antioxidant enzyme activities in the affected neurons. The ability of melatonin to shield these neurons from free radical damage is of particular interest since pyramidal neurons are also lost, via mechanisms involving oxidative and nitrosative stress, in a variety of neurodegenerative diseases. In cerebellar granule neurons, Dabbeni-Sala et al. [32] found that kainic acid increased mitochondrial free radical generation (identified with 2' ,7'-dichlorofluorescein) and impaired the function of complex II of the respiratory chain. Moreover, kainate promoted the activity of nitric oxide synthase thereby elevating the production of NO • and ultimately of ONOO-. In this series of in vitro studies, melatonin counter acted oxidative and nitrosative stress associated with the exposure of cerebellar granule neurons to KA.
The authors of the present study have reported that KA induces ROS and RNS production in splenic lymphocytes and enriched T lymphocytes. MLT proved effective in combating both the oxidative and nitrosative stress associated with the exposure of splenic lymphocytes and enriched T lymphocytes to KA.
Further, studies on C57BL/6 mice lacking specific T cell populations (CD4, CD8, and CD4/CD8 cells) and B cells [Igh-6(-/-)] demonstrated that influence of the adaptive immune response on KA-induced hippocampal neurodegeneration and suggest that B cell and T cell subsets may contribute differently to the pathogenesis induced by KA [10].
Confirmation of mitochondrial level of action of melatonin during excitotoxicity was provided by the observations that melatonin also prevented mitochondrial DNA damage [10,32] while also reducing seizures that often accompany kainic acid administration [33]. Neuronal death that occurs in these situations is presumably the result of apoptosis.
Chuang et al. [34] provided direct evidence that in fact apoptosis was the process that leads to cellular implosion after kainate is given. Three days following the intrastriatal injection of kainic acid, these workers noted the dramatic cytotoxic action of the drug along with elevated Bcl-2 immunoreactivity in TUNEL-positive cells. Although detectable, less severe damage was seen in the ipsilateral substantia nigra. Melatonin reduced Bcl-2 expression and preserved neuronal viability (reduced the number of TUNEL-positive neurons). Similar effects of melatonin on Bcl-2 expression in kainic acid-treated rat hippocampal neurons were reported by Yalcin and colleagues [35]. In addition to reducing hippocampal neuronal death due to its direct scavenging activity, melatonin may be protective since it stimulates the antioxidative enzymes, SOD and catalase, in these cells as well [36]. CD4/CD8-deficiency and increased age prevented KA-induced increase of both locomotor and rearing activities. The results suggest that a decline of immunological function is associated with aging, and both of them may contribute to the relative resistance to KA-induced neurotoxicity [37].
Chung and Han [10] surmised that the toxicity of kainic acid in relation to selective neuronal loss in the hippocampus may involve activation of microglia. Thus, besides enhancing the release of excessive amounts of the excitatory amino acid neurotransmitter, glutamate, which leads to damage of the post synaptic neuron, activates microglial cells and the production of ROS which would further exaggerate the neuronal damage kainic acid may also induce oxidative and nitrosative stress on splenic lymphocytes and enriched T lymphocytes. Also, antioxidant properties of exogenous melatonin can be a remedy to this effect of KA on lymphocytes.

Conclusion
The study clearly depicts that KA induces oxidative damage to splenic lymphocytes and enriched T lymphocytes in the form of apoptosis and inflammation which was evident by expression of various cytokines and spleen DNA integrity. This may lead to mutations and altered protein synthesis in due course of time. It is of interest to note that MLT has a protective action on the damage induced by KA by protecting the lymphocytes from such alterations. Hence exogenous MLT can be used as a therapeutic agent against immune toxicity by scavenging the free radical production induced by KA.

Disclosures
The authors have no financial conflict of interest.