Tapan K. Mondal

Autoantibody-mediated neuroinflammation: pathogenesis of neuropsychiatric systemic lupus erythematosus in the NZM88 murine model.

Autoantibodies play an important role in central nervous system manifestations of neuropsychiatric systemic lupus erythematosus (NPSLE). Previous studies have shown that the lupus-prone NZM88 strain has major neural deficits and high titers of serum IgG to brain antigens. ELISA was performed to detect the presence of IgG in different brain regions of NZM88 mice and to compare the levels with NZM2758 mice and control strains (NZW and BALB/c). There was a substantial increase of IgG in the substantia nigra (SN) and hypothalamus (HT) of brains from NZM88 mice compared to control NZW and BALB/c mice, whereas NZM2758 mice had more IgG in the cortex. The increased presence of IgG in the NPSLE-prone NZM88 mouse brain was paralleled by increased TNF-alpha and IL-12 in the SN and HT regions; significantly elevated expression of MHC Class-II was also observed in the SN of NZM88 mice and cortex of NZM2758 mice. A co-culture system of dopaminergic neurons and microglia was used to demonstrate that NZM88 sera modifies dopaminergic cell activity only in the presence of microglia and that TNF-alpha is synthesized and released in this co-culture. This study demonstrates a functional link between the autoantibodies, the activation of microglia, and neuronal function associated dopamine production, which is suggested to be causally related to the predominant NPSLE syndromes.

Serum antibodies from Parkinson's disease patients react with neuronal membrane proteins from a mouse dopaminergic cell line and affect its dopamine expression

Evidence exists suggesting that the immune system may contribute to the severity of idiopathic Parkinsons disease (IPD). The data presented here demonstrates that antibodies in the sera of patients with IPD have increased binding affinity to dopaminergic (DA) neuronal (MN9D cell line) membrane antigens in comparison to antibodies in sera from healthy controls. In general, the degree of antibody reactivity to these antigens of the mouse MN9D cell line appears to correlate well with the disease severity of the IPD patients contributing sera, based on the total UPDRS scores. Surprisingly, the sera from IPD patients enhanced the DA content of MN9D cells differentiated with n-butyrate; the n-butyrate-differentiated MN9D cells had a greater concentration of DA (DA/mg total protein) than undifferentiated MN9D cells, especially early in culture. Although the IPD sera did not directly harm MN9D cellular viability or DA production, in the presence of the N9 microglial cell line, the amount of DA present in cultures of untreated or n-butyrate-treated MN9D cells was lowered by the IPD sera. The results suggest the involvement of antibodies in the decline of dopamine production and, thus, the potential of immune system participation in IPD.

Reduced glutathione is highly expressed in white matter and neurons in the unperturbed mouse brain — Implications for oxidative stress associated with neurodegeneration

Oxidative stress is implicated in the pathogenesis of many neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. The depletion of glutathione (GSH) a powerful antioxidant renders cells particularly vulnerable to oxidative stress. Isolated neuronal and glial cell culture studies suggest that glia rather than neurons have greatest reserves of GSH, implying that neurons are most sensitive to oxidative stress. However, pathological in vivo studies suggest that GSH associated enzymes are elevated in neurons rather than astrocytes. The active, reduced form of GSH is rapidly degraded thus making it difficult to identify the location of GSH in post-mortem tissue. Therefore, to determine whether GSH is more highly expressed in neurons or astrocytes we perfused mouse brains with a solution containing NEM which reacts with the sulfhydryl group of GSH, thus locking the active form in situ, prior to immunostaining with an anti-GS-NEM antibody. We obtained brightfield and fluorescent digital images of sections stained with DAPI and antibodies directed against GS-NEM, glial fibrillary acidic protein (GFAP) in regions containing the hippocampus, striatum, frontal cortex, midbrain nuclei, cerebellum and reticular formation neurons. GSH was most abundant in neurons and white matter in all brain regions, and only in occasional astrocytes lining the third and fourth ventricles. High levels of GSH in neurons and white matter, suggests astrocytes rather than neurons may be particularly vulnerable to oxidative stress.

Mercury impairment of mouse thymocyte survival in vitro: involvement of cellular thiols.

Heavy metals are well known to be able to induce immunotoxicity, but comparative metal studies related to apoptosis have not been conducted. In the present study, the effects of arsenic, cadmium, gold, lead, manganese, and mercury on thymocytes from BALB/c mice were analyzed. Thymic cells were cultured for 3–24 h in vitro in the absence or presence of metal, and markers of apoptosis or cell death, including annexin V binding, DNA loss/oligonucleosomal fragmentation, 7-amino-actinomycin D uptake (loss of impermeance), changes of the mitochondrial membrane potential (JC-1 fluorescence), and Western analysis of cellular thiols, were assayed. Mercury (Hg) was the only metal shown to be consistently toxic with the dose and times utilized. Cadmium (Cd) was the only other metal tested that also produced some significant level of DNA loss; however, the induction of apoptosis by Cd was not as consistent as that observed with Hg. When Hg was added with 2-mercaptoethanol (2-ME), Hg produced greater toxicity. Endogenous DNA synthesis by thymocytes was immediately inhibited by Hg and Hg + 2-ME. The Hg + 2-ME-induced apoptosis appeared to be associated with altered levels of cellular thiols, in that glutathione (GSH) depletion was significant in comparison to the non-metal control and Hg alone. The increased Hg-induced toxicity in the presence of 2-ME likely was due to the ability of 2-ME to enhance (10- to 20-fold) the cellular uptake of Hg. Western analysis with biotin maleimide demonstrated that Hg + 2-ME and to a lesser extent the positive control dexamethasone eliminated many reactive thiols; the major thiol-reactive protein still reactive with the maleimide probe had an approximate Molecular Mass of 45 kD. Surprisingly, Hg alone enhanced the expression of this thiol-expressing protein, which by Mass Spectrometry (MS)/MS analysis was shown to be β-actin. Hg also produced the appearance of yet to be identified new proteins. Based on the results with Hg + 2-ME, it is suggested that numerous protein thiols participate in maintenance of cell survival and their loss is associated with apoptosis. The increased expression of new thiol-reactive proteins or thiol-reactive proteins with altered electrophoretic profiles needs to be further investigated. However, the enhanced toxicity attributed to Hg + 2-ME suggests that increased intracellular oxidative stress, observed as increased depletion of GSH, is responsible for the accelerated cell death. This work was supported by National Institutes of Health grant ES11135. The authors acknowledge use of the Immunology and Biological Mass Spectrometry Facilities of Wadsworth Center. We also especially appreciate the assistance of Kathy Lubowski, Joan Pedersen-Lane, and Renjie Song.

Detection of Immunoglobulin Isotypes from Dried Blood Spots

The study was designed to determine the sensitivity and reproducibility of recovering immunoglobulin (Ig) isotypes (IgG subclasses, IgA, IgE and IgM classes) from dried blood spots (DBS), a methodologic subcomponent of the Upstate KIDS Study. A multiplexed Luminex assay was used for IgG1/2/3/4, IgA and IgM analysis; an ELISA was used for IgE. Plasma samples from de-identified patients were used to compare the Luminex assay with nephelometry, which is routinely used to quantify IgA, IgG and IgM in clinical samples. The IgE ELISA was compared to an immunofluorescence assay. Prior to evaluation of punches from newborn dried blood spots (NDBSs), recoveries of Ig from punches of cord blood DBSs (CBDBSs) vs. plasma from the same cord bloods were compared. Although the recoveries of Ig from plasma and DBSs were not comparable, which could be due to cell lysates in the DBS samples, the analyses were reproducible. Additionally, the levels of IgA, IgG2, IgG4, and IgM recovered from CBDBSs positively correlated with those in plasma. The DBS data is a relative value since it is not equivalent to the plasma concentration. The majority of Ig concentrations recovered from 108 newborns of the Upstate KIDs Study were within the range of newborn plasma Ig levels with the exception of IgG3. The IgG4 values displayed the greatest variance with a wide range (0.01-319 mg/dl), whereas, IgG1 values had the narrowest range (85.2-960.4 mg/dl).

Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3+ (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19+ B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.

Sex effects of interleukin-6 deficiency on neuroinflammation in aged C57Bl/6 mice.

High levels of Interleukin-6 (IL-6) are associated with an increased risk of dementia in the elderly and can increase neuroinflammation in mice. Dementia is more frequent in females, and IL-6 is regulated by estrogen, suggesting that elevated IL-6 levels may contribute to neuroinflammation and dementia particularly in women. Therefore we hypothesized that IL-6 deficient ((-/-)) female mice would have lower aging-related neuroinflammation than wild type (WT). We quantified neuroinflammatory markers which are affected by aging, and regulated by both estrogen and IL-6; glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), interferon gamma (IFNgamma), lipid peroxidation (MDA), and synaptic density (SNAP25) and in IL-6(-/-) and WT C57Bl/6 mice. To determine age effects we used mid-age (18months) and old-age (24months) mice, and to determine region specific effects we used the hippocampus which is impaired in dementia and the cerebellum which is unimpaired in dementia. Unexpectedly, there were no effects of IL-6 deficiency on GFAP, MDA or SNAP25 levels in females, but IL-6 deficiency was associated with lower cerebellar MBP (p<0.05) levels. Interestingly, the old-aged IL-6(-/-) males had higher GFAP and MDA levels (p<0.05) in both the hippocampus and cerebellum, in addition to a greater body weight than WT. We suggest that IL-6 is important for promoting myelin synthesis in aged females, and that drugs which inhibit the synthesis of IL-6 in males may inadvertently affect fatty acid metabolism and augment aging-related neuroinflammation.

Metallothionein differentially affects the host response to Listeria infection both with and without an additional stress from cold-restraint

Acute stress alters anti-bacterial defenses, but the neuroimmunological mechanisms underlying this association are not yet well understood. Metallothionein (MT), a cysteine-rich protein, is a stress response protein that is induced by a variety of chemical, biological, and psychological stressors, and MT has been shown to influence immune activities. We investigated MT’s role in the management of anti-bacterial responses that occur during stress, using a C57BL/6 (B6) strain that has targeted disruptions of the Mt1 and Mt2 genes (B6-MTKO), and a B6 strain that has additional copies of Mt (B6-MTTGN). The well-characterized listeriosis model was used to examine immune mechanisms that are altered by a 1-h stress treatment (cold-restraint, CR) administered just prior to bacterial infection. Intriguingly, MT gene doses both greater and lower than that of wild-type (WT) B6 mice were associated with improved host defenses against Listeria monocytogenes (LM). This augmented protection was diminished by CR stress in the MTKO mice, but transgenic mice with additional MT copies had no CR stress-induced increase in their listerial burden. During the transition from innate to adaptive immunity, on day 3 after infection, oxidative burst and apoptosis were assessed by flow cytometric methods, and cytokine transcription was measured by real-time quantitative PCR. MT gene expression and CR-stress affected the expression of IL-6 and TNFα. Additionally, these genetic and environmental modulations altered the generation of ROS responses as well as the number of apoptotic cells in livers and spleens. Although the level of MT altered the listerial response, MT expression was equally elevated by listerial infection with or without CR stress. These results indicate the ability of MT to regulate immune response mechanisms and demonstrate that increased amounts of MT can eliminate the immunosuppression induced by CR.

A physical/psychological and biological stress combine to enhance endoplasmic reticulum stress

The generation of an immune response against infectious and other foreign agents is substantially modified by allostatic load, which is increased with chemical, physical and/or psychological stressors. The physical/psychological stress from cold-restraint (CR) inhibits host defense against Listeria monocytogenes (LM), due to early effects of the catecholamine norepinephrine (NE) from sympathetic nerves on β1-adrenoceptors (β1AR) of immune cells. Although CR activates innate immunity within 2h, host defenses against bacterial growth are suppressed 2-3 days after infection (Cao and Lawrence 2002). CR enhances inducible nitric oxide synthase (iNOS) expression and NO production. The early innate activation leads to cellular reduction-oxidation (redox) changes of immune cells. Lymphocytes from CR-treated mice express fewer surface thiols. Splenic and hepatic immune cells also have fewer proteins with free thiols after CR and/or LM, and macrophages have less glutathione after the in vivo CR exposure or exposure to NE in vitro. The early induction of CR-induced oxidative stress elevates endoplasmic reticulum (ER) stress, which could interfere with keeping phagocytized LM within the phagosome or re-encapsuling LM by autophagy once they escape from the phagosome. ER stress-related proteins, such as glucose-regulated protein 78 (GRP78), have elevated expression with CR and LM. The results indicate that CR enhances the unfolded protein response (UPR), which interferes with host defenses against LM. Thus, it is postulated that increased stress, as exists with living conditions at low socioeconomic conditions, can lower host defenses against pathogens because of oxidative and ER stress processes.

Participation of liver stem cells in cholangiocarcinogenesis after aflatoxin B1 exposure of glutathione S-transferase A3 knockout mice:

Aflatoxin B1, arguably the most potent human carcinogen, induces liver cancer in humans, rats, trout, ducks, and so on, but adult mice are totally resistant. This resistance is because of a detoxifying enzyme, mouse glutathione S-transferase A3, which binds to and inactivates aflatoxin B1 epoxide, preventing the epoxide from binding to DNA and causing mutations. Glutathione S-transferase A3 or its analog has not been detected in any of the sensitive species, including humans. The generation of a glutathione S-transferase A3 knockout (represented as KO or -/-) mice has allowed us to study the induction of liver cancer in mice by aflatoxin B1. In contrast to the induction of hepatocellular carcinomas in other species, aflatoxin B1 induces cholangiocarcinomas in GSTA3-/- mice. In other species and in knockout mice, the induction of liver cancer is preceded by extensive proliferation of small oval cells, providing additional evidence that oval cells are bipolar stem cells and may give rise to either hepatocellular carcinoma or cholangiocarcinoma depending on the nature of the hepatocarcinogen and the species of animal. The recent development of mouse oval cell lines in our laboratory from aflatoxin B1-treated GSTA3-/- mice should provide a new venue for study of the properties and potential of putative mouse liver stem cells.