M. Kerry O'Banion

The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective

Elevation of the proinflammatory cytokine Interleukin-1 (IL-1) is an integral part of the local tissue reaction to central nervous system (CNS) insult. The discovery of increased IL-1 levels in patients following acute injury and in chronic neurodegenerative disease laid the foundation for two decades of research that has provided important details regarding IL-1s biology and function in the CNS. IL-1 elevation is now recognized as a critical component of the brains patterned response to insults, termed neuroinflammation, and of leukocyte recruitment to the CNS. These processes are believed to underlie IL-1s function in the setting of acute brain injury, where it has been ascribed potential roles in repair as well as in exacerbation of damage. Explorations of IL-1s role in chronic neurodegenerative disease have mainly focused on Alzheimer disease (AD), where indirect evidence has implicated it in disease pathogenesis. However, recent observations in animal models challenge earlier assumptions that IL-1 elevation and resulting neuroinflammatory processes play a purely detrimental role in AD, and prompt a need for new characterizations of IL-1 function. Potentially adaptive functions of IL-1 elevation in AD warrant further mechanistic studies, and provide evidence that enhancement of these effects may help to alleviate the pathologic burden of disease.

Interleukin‐1β Induces Prostaglandin G/H Synthase‐2 (Cyclooxygenase‐2) in Primary Murine Astrocyte Cultures

Abstract: Activation of glial cells and the consequent release of cytokines, proteins, and other intercellular signaling molecules is a well‐recognized phenomenon in brain injury and neurodegenerative disease. We and others have previously described an inducible prostaglandin G/H synthase, known as PGHS‐2 or cyclooxygenase‐2, that is up‐regulated in many cell systems by cytokines and growth factors and down‐regulated by glucocorticoid hormones. In cultured mouse astrocytes we observed increased production of prostaglandin E2 (PGE2) after stimulation with either interleukin‐1β (IL‐1β) or the protein kinase C activator phorbol 12‐myristate 13‐acetate (TPA). This increase in PGE2 content was blocked by pretreatment with dexamethasone and correlated with increases in cyclooxygenase activity measured at 4 h. Northern blots revealed concomitant increases in PGHS‐2 mRNA levels that peaked at 2 h and were dependent on the dosage of IL‐1β. Dexamethasone inhibited this induction of PGHS‐2 mRNA by IL‐1β. TPA, basic fibroblast growth factor, and the proinflammatory factors tumor necrosis factor α and lipopolysaccharide, but not interleukin‐6, also stimulated PGHS‐2 mRNA expression. Relative to IL‐1β, the greater increases in PGE2 production and cyclooxygenase activity caused by TPA correlated with a greater induction of PGHS‐2 mRNA. Furthermore, NS‐398, a specific inhibitor of cyclooxygenase‐2, blocked >80% of the cyclooxygenase activity in TPA‐treated astrocytes. These findings indicate that increased expression of PGHS‐2 contributes to prostaglandin production in cultured astrocytes exposed to cytokines and other factors.

Chronic Interleukin-1β Expression in Mouse Brain Leads to Leukocyte Infiltration and Neutrophil-Independent Blood–Brain Barrier Permeability without Overt Neurodegeneration

The proinflammatory cytokine interleukin-1β (IL-1β) plays a significant role in leukocyte recruitment to the CNS. Although acute effects of IL-1β signaling in the mouse brain have been well described, studies elucidating the downstream effects of sustained upregulation have been lacking. Using the recently described IL-1βXAT transgenic mouse model, we triggered sustained unilateral hippocampal overexpression of IL-1β. Transgene induction led to blood–brain barrier leakage, induction of MCP-1 (monocyte chemoattractant protein 1) (CCL2), ICAM-1 (intercellular adhesion molecule 1), and dramatic infiltration of CD45-positive leukocytes comprised of neutrophils, T-cells, macrophages, and dendritic cells. Despite prolonged cellular infiltration of the hippocampus, there was no evidence of neuronal degeneration. Surprisingly, neutrophils were observed in the hippocampal parenchyma as late as 1 year after transgene induction. Their presence was coincident with upregulation of the potent neutrophil chemotactic chemokines KC (keratinocyte-derived chemokine) (CXCL1) and MIP-2 (macrophage inflammatory protein 2) (CXCL2). Knock-out of their sole receptor CXCR2 abrogated neutrophil infiltration but failed to reduce leakage of the blood–brain barrier.

TNFα and IL-1β mediate intercellular adhesion molecule-1 induction via microglia–astrocyte interaction in CNS radiation injury

Radiation injury to the central nervous system (CNS) results in glial activation accompanied by expression of pro-inflammatory cytokines and adhesion molecules. In this study we demonstrate intercellular adhesion molecule-1 (ICAM-1) induction in the irradiated mouse brain at the mRNA and protein levels. Immunocytochemical analysis revealed that ICAM-1 protein was primarily expressed in endothelial cells and microglia. In vitro, ionizing radiation significantly induces TNF alpha, IL-1beta and ICAM-1 mRNA in primary microglia cultures. Interestingly, although ionizing radiation activated primary astrocyte cultures, it did not induce ICAM-1 expression. However, exposure of astrocytes to conditioned medium collected from irradiated microglia resulted in ICAM-1 induction, which was abrogated when the conditioned medium was pre-incubated with neutralizing antibodies raised against murine TNF alpha and IL-1beta. These results indicate that pro-inflammatory cytokines may be necessary for ICAM-1 expression in astrocytes in CNS radiation injury.

Enhanced glial activation and expression of specific CNS inflammation-related molecules in aged versus young rats following cortical stab injury

Aging is associated with increased glial responsiveness that may enhance the brains susceptibility to injury and disease. To determine whether unique age-related molecular responses occur in brain injury, we assessed mRNA levels of representative central nervous system (CNS) inflammation-related molecules in young (3 months) and aged (36 months) Fisher 344/Brown Norwegian F1 hybrid rats following cortical stab. Enhanced glial activation in older animals was accompanied by increased expression of a subset of inflammation-related mRNAs, including IL-1beta, TNFalpha, IL-6, ICAM-1, inducible nitric oxide synthase (iNOS), metalloproteinase-9 (MMP-9), and complement 3alpha-chain 1 (C3alpha1). Recognition of these age-specific differences may guide development of novel treatment regimes for older individuals.

Noradrenergic depletion increases inflammatory responses in brain: effects on IκB and HSP70 expression

The inflammatory responses in many cell types are reduced by noradrenaline (NA) binding to β‐adrenergic receptors. We previously demonstrated that cortical inflammatory responses to aggregated amyloid beta (Aβ) are increased if NA levels were first depleted by lesioning locus ceruleus (LC) noradrenergic neurons, which replicates the loss of LC occurring in Alzheimers disease. To examine the molecular basis for increased responses, we used the selective neurotoxin DSP4 to lesion the LC, and then examined levels of putative anti‐inflammatory molecules. Inflammatory responses were achieved by injection of aggregated Aβ1–42 peptide and IL‐1β into frontal cortex, which induced neuronal inducible nitric oxide synthase (iNOS) and microglial IL‐1β expression. DSP4‐treatment reduced basal levels of nuclear factor kappa B (NF‐κB) inhibitory IκB proteins, and of heat shock protein (HSP)70. Inflammatory responses were prevented by co‐injection (ibuprofen or ciglitzaone) or oral administration (pioglitazone) of peroxisome proliferator‐activated receptor gamma (PPARγ) agonists. Treatment with PPARγ agonists restored IκBα, IκBβ, and HSP70 levels to values equal or above those observed in control animals, and reduced activation of cortical NF‐κB. These results suggest that noradrenergic depletion reduces levels of anti‐inflammatory molecules which normally limit cortical responses to Aβ, and that PPARγ agonists can reverse that effect. These findings suggest one mechanism by which PPARγ agonists could provide benefit in neurological diseases having an inflammatory component.

Cyclooxygenase Inhibition as a Strategy to Ameliorate Brain Injury

Cyclooxygenase (COX) is the obligate, rate-limiting enzyme for the conversion of arachidonic acid into prostaglandins. Two COX enzymes have been identified: a constitutively expressed COX-1 and an inducible, highly regulated COX-2. Widely used to treat chronic inflammatory disorders, COX inhibitors have shown promise in attenuating inflammation associated with brain injury. However, the use of COX inhibition in the treatment of brain injury has met with mixed success. This review summarizes our current understanding of COX expression in the central nervous system and the effects of COX inhibitors on brain injury. Three major targets for COX inhibition in the treatment brain injury have been identified. These are the cerebrovasculature, COX-2 expression by vulnerable neurons, and the neuroinflammatory response. Evidence suggests that given the right treatment paradigm, COX inhibition can influence each of these three targets. Drug interactions and general considerations for administrative paradigms are also discussed. Although therapies targeted to specific prostaglandin species, such as PGE2, might prove more ameliorative for brain injury, at the present time non-specific COX inhibitors and COX-2 specific inhibitors are readily available to researchers and clinicians. We believe that COX inhibition will be a useful, ameliorative adjunct in the treatment of most forms of brain injury.

Cranial Irradiation Leads to Acute and Persistent Neuroinflammation with Delayed Increases in T-Cell Infiltration and CD11c Expression in C57BL/6 Mouse Brain

Radiotherapy is commonly employed to treat cancers of the head and neck and is increasingly used to treat other central nervous system (CNS) disorders. Exceeding the radiation tolerance of normal CNS tissues can result in sequelae contributing to patient morbidity and mortality. Animal studies and clinical experience suggest that neuroinflammation plays a role in the etiology of these effects; however, detailed characterization of this response has been lacking. Therefore, a dose–time investigation of the neuroinflammatory response after single-dose cranial irradiation was performed using C57BL/6 mice. Consistent with previous reports, cranial irradiation resulted in multiphasic inflammatory changes exemplified by increased transcript levels of inflammatory cytokines, along with glial and endothelial cell activation. Cranial irradiation also resulted in acute infiltration of neutrophils and a delayed increase in T cells, MHC II-positive cells, and CD11c-positive cells seen first at 1 month with doses ≥15 Gy. CD11c-positive cells were found almost exclusively in white matter and expressed MHC II, suggesting a “mature” dendritic cell phenotype that remained elevated out to 1 year postirradiation. Our results indicate that cranial irradiation leads to persistent neuroinflammatory changes in the C57BL/6 mouse brain that includes unique immunomodulatory cell populations.

Prostaglandin G/H Synthase-2 (Cyclooxygenase-2) mRNA Expression is Decreased in Alzheimer's Disease

Recent evidence suggests that the use of nonsteroidal anti-inflammatory drugs (NSAIDS) is beneficial for therapy or prevention of Alzheimers disease (AD). The major anti-inflammatory action of NSAIDS is to inhibit prostaglandin G/H synthase-2 (PGHS-2), the first committed enzymatic step for prostaglandin biosynthesis. We have previously shown that PGHS-2 message is induced by Interleukin-1 beta and other inflammatory mediators in primary cultures of rodent astrocytes. To determine whether similar elevations of PGHS-2 occur as part of the gliosis in AD, we quantified PGHS-2 mRNA levels in control and AD brain by Northern hybridization analysis. To our surprise we found that PGHS-2 mRNA levels were reduced threefold in AD neocortex relative to control brain tissue. In contrast, levels were not reduced in putamen, an area that is relatively spared in AD. To localize PGHS-2 mRNA production in control and AD brain, sections of neocortex and hippocampus were hybridized with a 35S-labeled riboprobe for human PGHS-2 followed by immunocytochemistry with antibodies against neuron specific enolase (NSE) or glial fibrillary acidic protein (GFAP). Our findings indicate that PGHS-2 message is primarily localized to cells that stain for NSE rather than GFAP. Furthermore, in the three cases we examined, PGHS-2 hybridization per neuron appeared to be reduced in AD. Thus, the decrease we observe in overall PGSH-2 mRNA levels is likely to reflect both the known decline in numbers of neurons in AD as well as a lowered capacity for neuronal synthesis of PGHS-2, perhaps due to dysfunction or a loss of synaptic input.

Adenovirus-Mediated Transgene Expression in Nonhuman Primate Brain

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.