CNS Cytokines 365 However, TGFβ1 can also behave in a proinflammatory manner (Grammas and Ovase, 2002). With murine autoimmune encephalomyelitis, overproduction of TGFβ1 locally in the brain led to a more severe and earlier onset of the disease with increased infiltration of mononuclear cells in the brain (Luo et al., 2007). TGFβ1 along with IL-6 are known to promote IL-17 producing T cells, which enhance inflammation. The encephalomyelitis model seems to indicate that the brain will signal to the immune system in certain circumstances as was suggested by increased plasma levels of IL-1, IL-6, and α-1-antichymotrypsin in patients with Alzheimer’s disease (Licastro et al., 2000). In astrocyte cultures, TGFβ1, in the presence of LPS and IFN-γ, increased the expression of iNOS (NOS-2). It was determined that the increase in NOS-2 was due to enhanced proliferation of astrocytes producing NOS-2 (Hamby et al., 2006). In a mouse model for cerebral palsy, it was observed that neuronal-derived TGFβ1 mediated in some way the IL-9/mast cell interaction and exacerbated excitotoxicity in newborn mice (Patkai et al., 2001; Mesples et al., 2005). Further evidence of TGFβ1 involvement in mast cell activity comes from in vitro experiments with the mast cell line, D-36. In D-36 cultures, addition of TGFβ1 increased media histamine concentrations. It is the increase in the extracellular his- tamine concentrations that promotes the excitotoxic neuronal damage in cerebral palsy. Therefore, in neonatal cerebral palsy, the action of TGFβ1 on mast cells is proinflammatory in nature. 1.1.4 IL-33 and HMGB1 The cytokines IL-33 and HMGB1 are both present in the brain at relatively high concentration within the nucleus of cells, astrocytes in the case of IL-33 and neu- rons and oligo-dendrocyte–like cells for HMGB1(Hudson et al., 2008; Kim et al., 2008). HMGB1 is a nonhistone DNA-binding protein of the nucleoprotein complex, and it regulates TNF-α expression. HMGB1 also is able to increase proinflamma- tory cytokine levels by enhancing the signaling of TLR9 (Ivanov et al., 2007). Both proinflammatory proteins are released into the cytoplasm and subsequently secreted upon stimulation through signals stemming from infection or injury. Like IL-1β and IL-18, IL-33 is released by inflammasome activation of caspase-1 (Martinon et al., 2007); the central components of inflammasomes detect cytosolic microbial components and “danger” signals, such as ATP and toxins. Glial and astrocyte- enhanced cultures stimulated with PAMPs and ATP produced elevated levels of IL-1β and IL-33 i n the culture media. Culture supernatants from these astrocyte enhanced cultures were able to induce the secretion of IL-6, IL-13, and MCP- 1 from a mast cell line, MC/9 in culture, in a similar fashion to the addition of IL-33 alone (Hudson et al., 2008). It was also noted that IL-33 levels were increased in brains of mice infected with Theiler’s murine encephalomyelitis virus with the promotion of various innate immune effectors on glial cells (Hudson et al., 2008). The proinflammatory effects of HMGB1 were studied in the postischemic brain of rats. Ischemia injury in the brain proceeds with excitotoxicity-induced acute neu- ronal cell death in the ischemic core, followed by delayed damage to the penumbra (Lee et al., 2000b). It was observed that HMGB1 was immediately released into 366 J. Kasten-Jolly and D.A. Lawrence the extracellular space after ischemia and subsequently promoted neuroinflamma- tion by microglial activation. Downregulation of HMGB1 expression via (sh) RNA decreased infarct size, microglial activation, and proinflammatory marker induc- tion (Kim et al., 2006). After 1 h of middle cerebral artery occlusion HMGB1 was immediately translocated from the neuron nuclei into the extracellular space during the excitotoxicity-induced acute damage process. About two days after reperfusion HMGB1 was notably induced in activated microglia, astrocytes, and microvas- cular structures. This induction of HMGB1 expression was sustained for several days. The results emphasize the paracrine and autocrine function of HMGB1 in the postischemic brain (Kim et al., 2008). 1.1.5 IL-10 and IL-13 Interleukins 10 and 13 are anti-inflammatory, because they inhibit IL-1β bioac- tivity. IL-10 is capable of directly blocking IL-1 expression (Wong et al., 1997), whereas IL-13 induces the synthesis of the IL-1 receptor antagonist (IL-1ra). These interleukins are considered CD4 + T-helper subset type 2 (Th2) and function to coun- teract the effects of CD4 + T-helper subset type 1 (Th1) proinflammatory activities by inhibiting their action (Martino et al., 2000). In general, cytokines produced by each Th cell subset are inhibitory for the opposite subset. Production of these Th2 cytokines during neuroinflammation may be through the appearance of brain- derived heat shock protein (HSP) peptide complexes in the periphery (Galazka et al., 2006). HSP-induced downregulation of immune responses may involve the genera- tion of immune cell subsets, such as Ag-specific Th2 cells, which secrete cytokines such as IL-10 that inhibit the proinflammatory process. Inhibition of inflamma- tory processes by IL-10 and IL-13 has been connected with ceramide production. Proinflammatory cytokines promote ceramide production through hydrolysis of plasma membrane sphingomyelin in brain cells. Ceramide plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The anti-inflammatory cytokines IL-10 and IL-13 are capable of blocking ceramide pro- duction through a mechanism involving activation of phosphatidylinositol-3-kinase. By blocking ceramide production IL-10 and IL-13 are able to inhibit apoptosis caused by the actions of proinflammatory cytokines, IL-1 and TNF-α (Pahan et al., 2000). 1.2 MAP Kinases and Stress Kinases Stress stimuli come in a variety of forms, such as deprivation of trophic factors, ionizing radiation, free radicals (e.g., peroxynitrite), hypoxia, ischemia, heat shock, lipid second messengers (such as ceramide) (Singh et al., 1998), TNF-α,orFas- ligand. In the brain, neurons are especially susceptible to stress stimuli; these stimuli lead to activation of intracellular pathways that either promote apoptosis or defense- adaptation mechanisms. At least three such pathways have been well studied. These pathways lead to the activation of c-Jun N-terminal kinases (JNKs), p38 kinases, CNS Cytokines 367 Fig. 2 Outline of signal transduction pathways for stress kinases, JNKs, and for Map kinases, p38 and ERK1/2. Shown is the cross-talk between the various pathways. Map kinases MEKK1/2/3 activate the MAP kinases MKK4 (JNKK1) and MKK7 (JNKK2). MKK4/7 can also be activated by the Map kinases TAK1 and Ask1, which can also activate MKK3/6 of the p38 s pathway. Activation of Rap-GTP is capable of activating the p38 and ERK1/2 pathways. As indicated, ceramide can activate the p38 pathway, and, also, has been suggested to play a role in activation of the ERK1/2 pathway and extracellular signal-regulated kinases (ERKs) (Mielke and Herdegen, 2000). The cascades for these pathways are overlapping in places, as shown in Fig. 2.Note that each pathway can be stimulated by TNF-α and both p38 and JNK cascades can be stimulated by ceramide. Activation of JNK or p38 kinases leads to upregulation or activation of a number of factors, including transcription factors (ATF2, CREB, ELK1, MEF2C, CHOP), translation factors (eIF4E), MAP kinase-associated pro- teins, heat shock protein (Hsp27), and phosphorylation of tau at position Ser422 (phosphorylated in neurofibrillary tangles, but not in normal brain). Stimulation of the JNK pathway leads to inactivation of NFAT4, glucocorticoid receptor, and Bcl2, an inhibitor of apoptosis. Involvement of JNK, p38, and ERK1/2 MAP kinases in neurodegeneration is fur- ther supported by studies demonstrating that inhibition of their activation reduces brain inflammation and neuron damage (Barone et al., 2001; Angstwurm et al., 2004). Moreover, modulation of the purinergic P2X 7 receptor by oxidized ATP (oxATP), during LPS activation of microglia, led to attenuation of inflammatory mediators, resulting in inactivation of the p38 and NF-kB pathways and increased neuronal survival (Ferrari et al., 2006; Choi et al., 2007). Neuronal damage via the p38 pathway can be modulated by the tyrosine phosphatase SHP2 (Chong et al., 2003). It was observed that neurons from mice deficient in SHP2 showed 368 J. Kasten-Jolly and D.A. Lawrence more increased susceptibility to damage by NO exposure than did their wild-type counterparts. SHP2 function was necessary for neuronal survival only after the induction of signal transduction pathways, such as p38, that would culminate in the cell’s death. In the absence of p38 activation, SHP2 remained dormant. In astro- cytes, the regulation of IL-6 expression is dependent upon activation of the p38 and ERK1/2 pathways and is modulated by oncostatin M (Van Wagoner et al., 2000). Aberrant expression of ICAM-1 on astrocytes during neuroinflammation will also result in the expression of IL-6 and other proinflammatory cytokines, IL-1α and IL-1β, through activation of the p38 and ERK1/2 pathways (Lee et al., 2000a). 1.3 Microglia Cells Microglial activation can enhance neuronal damage (Cunningham et al., 2002; Rivest, 2003). Injection of LPS into the hippocampus, cortex, and substantia nigra of rat brain resulted in high neurodegeneration in the substantia nigra, suggesting that this brain region possesses a high percentage of microglia cells (Kim et al., 2000); experiments described later support this distribution. In response to environmental toxins and certain endogenous proteins, microglia will become overactivated and will release reactive oxygen species (ROS), which can then cause neuronal dam- age (Block et al., 2007; Galea et al., 2003). Microglia cells are phagocytes and express a diverse array of membrane receptors (PRRs) that recognize a wide variety of molecular determinates, including phosphatidylserine (Li et al., 2003). Microglia receptors are constitutively expressed and bind PAMPS during the innate immune response. Prominent among these PRRs are the TLRs, of which microglia express TLRs 1–9. Other PRRs on microglia, include scavenger receptors, MAC1 recep- tors, and complexes of these receptors. The detection of extracellular superoxide is a common result of ligand recognition by PRRs and oxidative stress is a primary cause of neurodegeneration (Kifle et al., 1996). Through the activity of NADPH oxidase microglia become a robust source of free radicals, both extracellular and intracellular. Intracellular ROS provide a mechanism for proinflammatory signaling. Microglial NADPH oxidase has been associated with a number of neurodegenerative disorders, including Alzheimer’s disease and Parkinson’s disease (Kalaria et al., 1996). In addition to the extracellular effects, NADPH oxidase is crucial to microglia intracellular signaling. As an example, gangliosides activate microglia through a protein kinase C and NADPH oxidase mechanism (Farooqui et al., 2007). Gangliosides are able to bind to microglia cells via TLR4 (Jou et al., 2006). This binding signals through a JAK/STAT pathway and induces phosphorylation of STAT1 and STAT3 (Kim et al., 2002; Lee et al., 2005), which factors upregulate transcription of inflammation-associated genes (Heese et al., 1998; Pinteaux et al., 2007), such as iNOS, ICAM-1, and MCP-1. In general, the higher the intracellular ROS concentration is, the higher the inflammatory response will be in the microglia cells. However, prolonged ROS exposure will induce cumulative events harmful to the cell’s survival, such as lipid peroxidation and modification of proteins. Eventually the activated microglia CNS Cytokines 369 will become apoptotic themselves, an outcome that was shown to be enhanced by expression of B cell translocation gene-1 (BTG1) (Lee et al., 2003). Although the deleterious effects of activated and proliferating microglia cells are clear, there is some experimental evidence that suggests a protective role for these cells. In a mouse model for cerebral ischemia, ablation of proliferating resident microglia resulted in increased proinflammatory cytokine expression, increased infarct size, and a 2.7-fold increase in apoptotic cells, mostly neurons (Lalancette-Hebert et al., 2007). 1.4 Astrocytes Astrocytes, the most abundant glial cell of the CNS, can display innate immune responses triggered by a variety of insults (Xiao and Link, 1999; Becher et al., 2006). These cells possess a wide array of receptors including TLRs, nucleotide- binding oligomerization domains, double-stranded RNA-dependent protein kinase, scavenger receptors, mannose receptor, and components of the complement system (Bugno et al., 1999; Farina et al., 2007). Upon stimulation through one or more of these receptors, the cell will produce cytokines, IL-6, TGFβ,IFNβ,GM-CSF,BAFF, IL-1β, and TNF; chemokines, CCL2, CCL5, CCL20, CXCL-10, CXCL12, CXCL1, CXCL2, and CX3CL1; and neurotrophic factors, NGF, CNTF, BDNF, VEGF, IGF1, and LIF (Croll et al., 2004; Cotman et al., 2007; Krasowska-Zoladek et al., 2007). Two types of events result from astrocyte stimulation: activation of neighboring cells and further amplification of local innate immune responses and modification of BBB permeability and attraction of immune cells from the blood into the neu- ral tissue (Fitch and Silver, 1997; Andjelkovic and Pachter, 1998; Anthony et al., 1998; Stolp et al., 2005; Milner and Campbell, 2006; Andras et al., 2008). The lat- ter activity is mediated by chemokine expression (Mennicken et al., 1999; Biber et al., 2002; Hsieh et al., 2006). Migration of the astrocytes themselves can be induced by the presence of stromal cell-derived factor-1 (SDF-1) and upregulation of astrocyte CXCR4 by IL-6 plus cAMP (Odemis et al., 2002). Further evidence of the innate immune response activity of astrocytes stems from the cell’s abil- ity to secrete active α 1 -antichymotrypsin (Kanemaru et al., 1996) and expression of syncytin-1 in MS leading to the upregulation of iNOS through an old astrocyte specifically induced substance (OASIS) mechanism (Antony et al., 2007). Protective effects of astrocytes are mediated through the cell’s ability to release purines, adenosine and adenosine triphosphate, and guanosine and guanosine triphosphate (Ciccarelli et al., 2001). 1.5 Neuroinflammatory Aspects of Pb Toxicity 1.5.1 Pb Effects on Glial Cells In the CNS, Pb accumulates preferentially in glial cells rather than in neurons (Tiffany-Castiglioni et al., 1989; Lindahl et al., 1999), and Pb’s presence in these 370 J. Kasten-Jolly and D.A. Lawrence cells produces activities that are proinflammatory in nature. As indicated earlier, astrocytes may have a role in increasing permeability of the BBB during inflam- mation. It was observed that Pb was able to potentiate proinflammatory cytokine and glutamate-mediated increases in permeability of the BBB in mice (Dyatlov et al., 1998). In a study performed on Pb-exposed young (15–30-day-old) rats, it was observed that increased Pb levels resulted in astrocyte cell activation and pro- liferation, as indicated by elevated GFAP and S-100β in all brain regions examined (Struzynska et al., 2007). Results also showed increased production of IL-6 in the forebrain with a concomitant decrease in levels of the axonal markers synapsin-1 and synaptophysin. The study concluded that Pb caused chronic glial cell activation with coexisting inflammatory and neurodegenerative features. A more recent study using proton magnetic resonance to study the relative lev- els of certain metabolites in human brain regions indicated that Pb increased the myoinositol to creatine ratio (mI/Cr) in the hippocampus (Weisskopf et al., 2007). An increased mI/Cr is a distinctive aspect of Alzheimer’s disease and is thought to be indicative of gliosis. Therefore, the increased mI/Cr associated with increased bone Pb levels in humans is also suggestive of a neuroinflammatory aspect to Pb tox- icity. The study concluded that the glial effects observed might be the more sensitive indicators of the adverse effects of cumulative Pb exposure and these changes are similar to those seen in the early stages of Alzheimer’s disease. A further connection between Pb activity and neurodegenerative diseases is the ability of Pb to produce a deficiency in chaperone protein function which then compromises protein secre- tion, exacerbates protein aggregation, and increases sensitivity to oxidative stress. Alzheimer’s disease and Parkinson’s disease are characterized by a deficiency in the function of the chaperone protein GRP78 (Bip, HSPA5). In the absence of a diseased state GRP78 facilitates the maturation of the amyloid precursor protein and reduces or prevents the formation of extracellular amyloid deposits. Pb binds to GRP78 and can inhibit its function as a chaperone protein (White et al., 2007). Furthermore, Pb exposure of rodents from pnd0-pnd20 gave a transient increase in amyloid precur- sor protein mRNA synthesis (White et al., 2007). Therefore, Pb neurotoxicity shares several features of neurodegenerative disorders. 1.5.2 Pb Effects on Cytokines in the CNS It has been observed that exposure to the heavy metal, lead (Pb), can increase sus- ceptibility to infectious agents (Lawrence, 1981). Moreover, neonatal Pb exposure exacerbated sickness behavior in pups infected with Listeria monocytogenes; such sickness behavior was documented as loss of appetite and drinking, decreased body- weight gain, and lack of mobility (Dyatlov and Lawrence, 2002). These results suggested that Pb might modulate expression of proinflammatory cytokines in the brain. Gene expression of 14 cytokines was measured by real-time RT-PCR in the perfused brain tissue of male and female 21-day-old mouse pups. As shown ear- lier, there are no significant differences in the expression levels between males and females. Messenger RNA for IL-4, IL-10, and IL-12p40 was not detectable in the CNS Cytokines 371 Fig. 3 Pb effect on expression of cytokines IL-6, TGF-β1, and IL-18 in the brain. Cytokine mRNA from the brains of female and male mouse pups at pnd21 was quantified by real-time RT-PCR. Whole-brain RNA was isolated using a Qiagen Lipid Tissue kit. Mouse pup brains from each litter were pooled according to gender. All cytokine RNA quantitation results were normalized to endogenous GAPDH. Each bar represents mean ± S.D. for an N of 3 L. Significance, indicated by an asterisk, ∗ , was determined by the Student’s t-test, p < 0.05. The p value for both female and male IL-6 gene expression ±Pb is 0.056 and the p value for male TGF-β1 gene expression ±Pb is 0.20 (Data for IL-6 and TGF-beta has been published in Kasten-Jolly et al., J. Biochem. Molec. Toxicol. 2010) brains of these pnd21 mouse pups. Among the most abundant cytokine transcripts in the brain are those coding for IL-16, IL-15, IL-18, and TGF-β1. Exposure of the mouse pups to 0.1 mM Pb acetate from gd8 to pnd21 via the dam’s drinking water resulted in enhanced expression of IL-6 and TGF-β1 (Fig. 3). If Pb exposure was increased to 0.5 mM an increase in expression of IL-18 in whole brain tissue was observed for the female mouse pups at pnd21 (Fig. 3). Associated with the upregulation of proinflammatory cytokine gene expres- sion and generation of ROSs is the activation of stress kinases and MAP kinases. Microarray data from whole-brain RNA of 0.1 mM Pb-exposed (gd8 to pnd21) and unexposed control mice indicated upregulation of p38 and MAP kinases within the p38 cascade, such as MAPKAPK-2 (Table 1). The microarray data are in agree- ment with previous reports (Cordova et al., 2003; Leal et al., 2006) indicating that Pb exposure induces activation of the p38 and ERK1/2 MAP kinase pathways. The mechanism of Pb activation of these pathways is not yet completely understood, but it may occur through the generation of ROSs. 372 J. Kasten-Jolly and D.A. Lawrence Table 1 Effect of Pb on gene expression of map kinases a,d signal b ± S.D. GeneBank ID Gene Control Pb p-value c NM_011951 Mapk1 12,119 ± 719 12,998 ± 1,083 0.15 NM_015806 Mapk6 1485 ± 107 1655 ± 73 0.04 BC024684 Mapk11(p38) 256 ± 32 358 ± 52 0.02 AF128892 Mapk14 3350 ± 173 3638 ± 171 0.06 BM240207 Map2k4 5412 ± 244 5894 ± 358 0.06 AW541674 Map2k7 1157 ± 77 1232 ± 69 0.14 AA929089 Map3k7 3832 ± 167 4152 ± 401 0.14 BF166991 Map4k2 500 ± 29 583 ± 80 0.08 BB734681 Map4k5 363 ± 50 490 ± 90 0.05 NM_016713 Map4k6 2589 ± 64 3086 ± 86 0.005 BG918951 Mapkapk2 783 ± 20 994 ± 91 0.009 a Affymetrix MG430A GeneChip data. The data represent mouse brain total RNA from female mouse pups from 3 L of untreated control mice and 3 L of 0.1 mM Pb acetate (gd8 to pnd21) treated experimental mice. b Signal was normalized to the GAPDH signal on each respective GeneChip. c Statistics were performed by Student’s t-test, significant at p < 0.05. d Table has been published i n Kasten-Jolly et al., J. Biochem. Molec. Toxicol, 2010. One of the indicators of neuroinflammation or neurodegeneration is an increase in GFAP expression due to astrocyte activation and proliferation (Hauss-Wegrzyniak et al., 1998; Norris et al., 2005; O’Callaghan and Sriram, 2005; Pannu et al., 2005). Because Pb exposure seemed to enhance gene expression of IL-6 and TGF-β1, it was postulated that Pb might also increase the expression of GFAP. Both IL-6 and TGF-β signaling are needed to promote transcription of the GFAP gene (Taga and Fukuda, 2005). Microarray data for pnd21 female mouse pups exposed to 0.1 mM Pb (gd8-pnd21) showed that Pb did significantly increase gene expression of GFAP (Fig. 4), indicating that Pb may be promoting astrocyte activation. As indicated above, αMSH has an anti-inflammatory effect in that it can inhibit the degradation of IκBα; thereby blocking the activation of NFκB (a transcription factor for several proinflammatory cytokines). αMSH is a product of the POMC1 gene, which also codes for several other peptide hormones. The microarray data suggested that Pb could decrease POMC1 gene expression. Results obtained by real- time RT-PCR supported this finding (Fig. 5). Shown here are POMC1 RNA levels for untreated controls and Pb-treated females at pnd21 using two different concen- trations of Pb, 0.1 and 0.5 mM. As indicated, Pb dampened POMC1 gene expression at each concentration. Therefore, Pb may increase inflammation by decreasing the expression of αMSH through downregulation of the POMC1 gene. 1.6 Neuroinflammatory Effects of Metals Other than Pb Neurotoxicity of aluminum and copper are associated with the upregulation of stress-related gene expression patterns. Whole human genome microarray data CNS Cytokines 373 Fig. 4 Pb exposure enhances CNS expression of GFAP. Female BALB/c mice from 3 L treated or untreated with 0.1 mM Pb acetate from gd8 to pd21 via the dam’s drinking water, were sacrificed on day 21, and whole-brain RNA was isolated. GFAP expression was measured by Affymetrix GeneChip (MG 430A). Signal for GFAP was normalized to the signal for GAPDH on the same chip. Data were gathered from 3 control litters (distilled water) and 3 Pb-treated litters, that is, 6 GeneChips. The ∗ indicates a p < 0.05 for the difference in normalized signal between control and Pb-treated female mice Fig. 5 Pb effect on gene expression of POMC1. POMC1 mRNA from the brains of female mouse pups at pnd21 was quantified by real-time RT-PCR. Whole-brain RNA was isolated using a Qiagen Lipid Tissue kit. Female mouse pup brains from each litter were pooled during isolation of the RNA. POMC1 transcript quantitation was normalized against endogenous GAPDH. Each bar rep- resents the mean ± SEM for an N of 6 L for untreated controls and N = 3 L for each experimental group 374 J. Kasten-Jolly and D.A. Lawrence indicated that aluminum exposure of human neural cells in culture resulted in expression patterns similar to those seen in Alzheimer’s disease (Lukiw et al., 2005). Seven of the genes found to be highly upregulated were proinflammatory and were proapoptotic, such as NF-κB subunits, IL-1β precursor, cytosolic phos- phlipase A 2 , cyclooxygenase-2, beta-amyloid precursor protein (APP), and DAXX. Many of the genes upregulated by aluminum contained promoter binding sites for NF-κB or stress-inducible transcription factors, such as HIF-1, thereby suggesting a role for these two promoter binding factors in proinflammatory gene expression. An association between Al-induced neurotoxicity and Alzheimer’s disease was fur- ther supported by the finding that mice exposed to Al or Cu showed enhanced oxidative stress and an accumulation of amyloid β peptides (Becaria et al., 2006). Concomitant exposure of t he mice to both metals, Al and Cu, produced a coopera- tive effect on increasing APP levels. Another way of promoting neurodegeneration and altered organ development is by modifying the transcription of genes. This can be achieved by blocking transcription factor binding to the DNA. Sp1 is among the transcription factors affected by metal toxicity. Pb, Zn, and Cd modulate the bind- ing of Sp1 to its DNA target sequences. It was suggested that exposure to heavy metals could alter developmental gene expression in the brain through their interfer- ence with binding of Sp1 to its promoter sites (Zawia et al., 1998). A study of how Pb and other heavy metals inhibit transcription factors, like Sp1, was performed using synthetic peptides (Razmiafshari and Zawia, 2000). Here it was found that Pb and other metals, Zn, Cd, and Hg, formed complexes with the peptides that bound the double-stranded DNA with high affinity and did not allow Sp1 DNA- binding. This inhibition of Sp1 DNA binding occurred in a manner dependent on the metal/peptide complex concentration. Therefore, heavy metals can alter the activ- ity of DNA binding proteins and ultimately alter their function in terms of gene expression regulation. 2 Summary Neuroinflammation can be initiated by CNS entrance of immune cells, peripherally generated cytokines or cytokines produced by glial cells; neuroinflammation is char- acterized by increased concentrations of proinflammatory cytokines, chemokines, glial cell activation and proliferation, increased BBB permeability, and neuronal damage. The heavy metal Pb produces neural toxicity that has a number of factors in common with neuroinflammation, including increased GFAP levels, increased IL-6 expression, gliosis, increased BBB permeability, and decreased chaperone protein function. Other metals produce neurotoxicity similar to Pb with parallels to features of neurodegenerative disorders, such as Alzheimer’s and Parkinson’s diseases. Acknowledgments This work was supported by NIH grant # RO1 ES11135. We thank Michael Ryan and the late Robin Pietropaolo from the Wadsworth Center Microarray Core facility for their assistance in performing the microarray analysis. We thank Donghong Gao for her technical assistance with Pb acetate treating the mice through the dam’s drinking water and for obtaining the intact brain tissue for us. . by aluminum contained promoter binding sites for NF-κB or stress-inducible transcription factors, such as HIF-1, thereby suggesting a role for these two promoter binding factors in proinflammatory. membrane sphingomyelin in brain cells. Ceramide plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The anti -in ammatory cytokines IL-10 and. are proinflammatory in nature. As indicated earlier, astrocytes may have a role in increasing permeability of the BBB during in am- mation. It was observed that Pb was able to potentiate proinflammatory