Garth E. Ringheim

Interleukin-6 mRNA expression by cortical neurons in culture: Evidence for neuronal sources of interleukin-6 production in the brain

In this study, we investigated the capacity of murine cortical neurons to express interleukin-6 (IL-6) mRNA and protein in culture. Using in situ hybridization techniques, IL-6 mRNA was localized to neuronal cells in these cultures. Moreover, IL-6 mRNA expression as measured by in situ and PCR was shown to be upregulated by the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). This was consistent with the dose and time-dependent increases in IL-6 secreted protein observed from cultures stimulated with IL-1 beta and TNF-alpha. Taken together, the data suggest that neurons are capable of participating more directly in the CNS cytokine network than previously thought and may play an important role in the inflammatory response activities in the brain.

Amyloid-β peptide fragments p3 and p4 induce pro-inflammatory cytokine and chemokine production in vitro and in vivo

Alzheimers disease (AD) pathology is characterized by senile plaques containing amyloid‐β (Aβ) peptide, a protein with neurotoxic and glial immune activating potential. In addition to the highly amyloidogenic peptides Aβ(1–40/42), plaques contain amino‐terminal truncated Aβ peptides including the alpha secretase‐generated p3 fragments Aβ(17–40/42). In the present study, Aβ(17–40/42), Aβ(1–40/42), Aβ(1–16), and Aβ(25–35) aged in different solvents exhibited varying capacity to activate the murine microglia cell line MG‐7 depending on solvent, peptide ‘aging’, and peptide sequence that did not strictly correlate with β‐sheet formation. Aβ(17–40/42) or Aβ(1–42) stimulated production of the pro‐inflammatory cytokines interleukin (IL)‐1α, IL‐1β, IL‐6 and tumor necrosis factor‐α (TNF‐α), and the chemokine MCP‐1 from differentiated human monocytes (THP‐1) while little or no stimulation was observed with the other Aβ fragments. MG7 cells also produced these five pro‐inflammatory proteins in response to Aβ(1–42) whereas Aβ(17–40/42) elicited mainly TNF‐α and MCP‐1. Murine and human astrocyte cell lines (D30 and U373, respectively) were generally less responsive to Aβ fragments producing mainly IL‐6 and MCP‐1 in response to Aβ(1–42) or Aβ(17–40/42) fragments. In mice, an intracerebroventricular infusion of Aβ(1–42) significantly increased IL‐1α, IL‐1β, IL‐6 and MCP‐1 while Aβ(17–40/42) increased MCP‐1 and Aβ(17–40) increased IL‐1β. These results demonstrate that p3 and p4 Aβ fragments are pro‐inflammatory glial modulators and thus may play a role in development of the immunopathology observed in AD.

Enhancement of beta-amyloid precursor protein transcription and expression by the soluble interleukin-6 receptor/interleukin-6 complex

We investigated a potential role for the soluble interleukin-6 receptor (sIL-6R) in modulating interleukin-6 (IL-6) function in the central nervous system by assessing IL-6 and sIL-6R effects on beta-amyloid precursor protein (beta-APP) transcription and expression in cells of human neuronal origin. Cells transfected with a luciferase reporter plasmid containing a 3.8 kb DNA fragment of the beta-APP promoter were shown to have inducible promoter activity when treated with phorbol ester or basic fibroblast growth factor, but not when treated with lipopolysaccharide or Il-6. PCR amplification analysis revealed the presence of mRNA encoding the signaling subunit of the Il-6 receptor complex, the gp130 subunit, at levels approximating that found in human cortical tissue. The mRNA encoding the IL-6 receptor, however, was poorly expressed and was detectable only at high amplification cycles. When purified sIL-6R protein was added together with IL-6, there was a rapid induction of promoter activity within 2 h of stimulation followed by elevations in protein levels of both cell-associated and secreted beta-APP. Analysis of mRNA transcripts from human cortical brain tissue and cell cultures derived from fetal human brain demonstrated the presence of an alternatively spliced secreted form of the IL-6 receptor mRNA, suggesting that cells of the central nervous system may themselves be a source of sIL-6R protein. The capacity for sIL-6R to enhance IL-6 function and broaden the IL-6 target cell population in the brain has implications for the regulation of beta-APP expression in disease states such as Alzheimers disease where elevations in brain IL-6 levels have been reported.

IL-10 and glucocorticoids inhibit Aβ(1–42)- and lipopolysaccharide-induced pro-inflammatory cytokine and chemokine induction in the central nervous system

Alzheimers disease (AD) is characterized by neuropil threads composed of structurally abnormal neurites, neurons containing paired helical filaments of tau protein, and extracellular deposits of amyloid-beta (Abeta) peptide, a protein fragment having neurotoxic and glial immune response activating potential. In the present study, we demonstrate that an acute intracerebroventricular (icv) injection of Abeta(1-42) in the mouse induces a time- and dose-dependent production of IL-1alpha, IL-1beta, IL-6 and MCP-1 in the hippocampus and cortex as measured by ELISA. Cytokine and chemokine levels were maximal at 9 h, with MCP-1 and IL-1alpha remaining elevated over a 24 h period and IL-1beta remaining elevated over a 48 h period. The temporal profile of Abeta-induced cytokine induction differed from that observed for LPS. Following an icv injection of LPS, maximal levels of IL-1alpha, IL-1beta, IL-6 and MCP-1 were attained by 9 h and, except for MCP-1, returned to levels indistinguishable from control at 24 h. MCP-1 remained elevated at 24 h and returned to basal levels at 48 h. In contrast, little production of TNF-alpha was observed under either Abeta or LPS acute stimulus conditions. Treatment with anti-inflammatory agents such as prednisolone, dexamethasone, or IL-10 inhibited both Abeta- and LPS-induced cytokine and chemokine production in the brain. In summary, icv administration of Abeta and LPS induced elevated brain levels of pro-inflammatory cytokines that could be inhibited by immune suppressing drugs and anti-inflammatory proteins, thus providing support for the utility of anti-inflammatory therapeutics in modulating the immunopathology observed in brain inflammatory diseases such as AD.

ADDITIVE EFFECTS OF BASIC FIBROBLAST GROWTH FACTOR AND PHORBOL ESTER ON β-AMYLOID PRECURSOR PROTEIN EXPRESSION AND SECRETION

Expression of the beta-amyloid precursor protein (beta-APP), a proteoglycan whose proteolytically derived fragments have been implicated in the neuropathology observed in Alzheimers disease, is regulated by a variety of stimuli including cytokines, phorbol esters, and growth factors. In this study we report the effects of basic fibroblast growth factor (bFGF) and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on beta-APP expression and secretion in SKNMC human neuroblastoma cells. Treatment of the cells with bFGF for 24 h increased APP promoter activity 200%, cell-associated full-length protein 189%, and secreted amino-terminal fragments 192% compared to basal levels. Treatment of the cells with PMA for 24 h also up-regulated APP expression and secretion with increases of 170, 112, and 161% being observed for promoter activity, cell-associated full-length protein, and secreted amino-terminal fragments, respectively. The effects of bFGF and PMA on the expression and secretion of beta-APP were additive and distinct in that: (a) co-treatment of the cells with maximally stimulating doses of bFGF and PMA had an additive effect on both induced full-length protein expression (242%) and secretion of amino-terminal fragments (311%) compared to basal levels; (b) net levels of full-length protein expression and secretion induced by bFGF and PMA differed significantly from each other; and (c) down-regulation of phorbol ester-stimulated protein kinase C by pre-treatment of the cells for 24 h with 1 microM PMA failed to attenuate bFGF-induced transcription or induced secretion of beta-APP.

Glial Modulating and Neurotrophic Properties of Propentofylline and Its Application to Alzheimer's Disease and Vascular Dementia

Alzheimer’s disease (AD) and vascular dementia (VaD) are two of the most common forms of dementia in the elderly. Together they represent one of the largest unmet medical needs of the Western industrialized nations where population demographics indicate a trend towards declining birth rates along with an increase in life expectancy. Alzheimer’s disease is a progressive neurodegenerative condition that is associated with plaque deposits of beta amyloid (Aβ), tangled appearing dystrophic neurons, and pronounced cellular loss consisting primarily of neurons and glia. Vascular dementia, in contrast, is most often associated with stroke, lacunar infarcts, and associated white matter lesions. Though distinct in their pathological characteristics, it is not uncommon to find the white matter lesions associated with VaD occurring in clinically diagnosed AD patients.1,2 It is likely, therefore, that therapeutic treatments targeted primarily for either VaD or AD will have therapeutic benefit on the other condition depending on the extent to which the two pathologies coexist in an individual. It is also likely that a therapeutic agent will have benefit in both diseases if common underlying disease mechanisms are being addressed. For example, neuronal cell death resulting from stress conditions associated with VaD may differ from that associated with AD, yet cell loss from either might be prevented if expression of a particular neuronal support factor such as nerve growth factor is upregulated. Therapeutic approaches to treating dementia can be categorized into two main groups: symptomatic and disease modifying. Symptomatic treatments focus primarily on compensating for neuronal cell loss or functional impairment most often by seeking to enhance activity of the remaining neuronal cell populations. Examples of symptomatic treatments are acetylcholinesterase inhibitors that elevate the neurotransmitter acetylcholine or muscarinic agonists that enhance neurotransmitter receptor signaling. Disease modifying agents approach treatment as a matter of delaying disease progression by any one of several mechanisms that aim to ameliorate AD or VaD pathology. Treatment of the pathology would then result in the subsequent halting or slowing of further cognitive decline. One such approach is the

Cyclic AMP potentiation of cytokine-induced nitric oxide synthase activity in a murine astrocyte cell line

Astrocytes in culture have been previously shown to express inducible nitric oxide synthase (iNOS) following treatment with cytokines such as interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma). We report here on the effects of the cyclic nucleotide analogues 8-bromo-cyclic AMP and 8-bromo-cyclic GMP on cytokine-stimulated iNOS gene expression in a cultured murine astrocyte cell line. In these cells, neither 8-bromo-cyclic AMP nor 8-bromo-cyclic GMP alone was able to stimulate iNOS activity. Similarly, neither IL-1 beta nor IFN-gamma was capable of independently stimulating iNOS expression. Co-stimulation with both cytokines, however, resulted in measurable increases in iNOS activity, and correlated to increases in iNOS mRNA levels. The addition of 8-bromo-cyclic AMP, but not 8-bromo-cyclic GMP, was found to further enhance the expression of iNOS activity induced by IL-1 beta and IFN-gamma co-stimulation. This potentiation effect of 8-bromo-cyclic AMP correlated to a further elevation in iNOS mRNA levels over that produced by cytokine co-stimulation alone. However, 8-bromo-cyclic AMP co-treatment with either cytokine alone did not stimulate iNOS activity, indicating that the signal transduction pathway(s) involved in the potentiation effect of 8-bromo-cyclic AMP is functional only in the presence of both cytokines. These results indicate that cyclic AMP-mediated processes can participate in modulating the expression of astrocyte iNOS when the appropriate combinations of stimulatory cytokines are present.