Proteasome blockers inhibit TNF-alpha release by lipopolysaccharide stimulated macrophages and microglia: implications for HIV-1 dementia.

HIV-1 infection of the central nervous system can cause severe neurologic disease although only microglial cells and brain macrophages are susceptible to productive viral infection. Substances secreted by infected cells are thought to cause disease indirectly. Tumor necrosis factor alpha (TNF-alpha) is one candidate neurotoxin and is upregulated during HIV-1 infection of the brain, likely via activation of the transcription factor NF-kappaB. We used the proteasome inhibitors, MG132 and ALLN (N-acetyl-Leu-Leu-Norleucinal), to inhibit NF-kappaB activation in primary human fetal microglia (PHFM) and primary monocyte derived-macrophages, and showed that they could block TNF-alpha release stimulated by lipopolysaccharide (LPS) or TNF-alpha. In addition, we performed electrophoretic mobility shift analysis and determined that in microglia, the p50/p65 heterodimer of NF-kappaB is activated by LPS stimulation, and is inhibited by MG132. Thus, blockade of NF-kappaB activation in microglia in vitro can decrease production of TNF-alpha and may prove to be a novel strategy for treating HIV-1 dementia.

Tumor necrosis factor alpha inhibits glutamate uptake by primary human astrocytes. Implications for pathogenesis of HIV-1 dementia.

Human immunodeficiency virus (HIV) infection is commonly associated with neurological disease that occurs in the apparent absence of extensive infection of brain cells by HIV, suggesting that indirect mechanisms account for neuropathogenesis in the CNS, perhaps including changes in the normal neuroprotective functions of astrocytes. To test this hypothesis, we examined the effect of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFalpha), produced by HIV-1-infected macrophages and microglia, on glutamate transport by primary human fetal astrocytes (PHFAs). A dose-dependent inhibition of high affinity glutamate uptake sites was observed 12-24 h after addition of exogenous recombinant human TNFalpha to PHFAs. This effect was specific since it was blocked by a neutralizing monoclonal antibody directed against TNFalpha. Furthermore, the inhibitory effect was reproduced by a monoclonal antibody that is an agonist at the 55-kDa TNF receptor. These results suggest that the neurotoxic effects of TNFalpha may be due in part to its ability to inhibit glutamate uptake by astrocytes, which in turn may result in excitotoxic concentrations of glutamate in synapses.

Neuropathogenesis of AIDS.

Neurological disease directly attributable to HIV-1 infection (HIV dementia) is one of the most frequent disorders in persons with AIDS. HIV-1 dementia is associated with neuronal loss, but occurs in the absence of direct viral infection of neurons, suggesting that neurological damage occurs by an indirect mechanism. Recent studies have identified a number of candidate HIV-1 neurotoxins that may cause neuronal damage through common pathways involving the induction of oxidative stress and excitotoxicity. These findings suggest new therapeutic approaches to the prevention and treatment of HIV-1-induced neurological disease.

Tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant N-acetylcysteine and the genes bcl-2 and crmA.

Tumor necrosis factor alpha (TNF-alpha) is a candidate human immunodeficiency virus type 1-induced neurotoxin that contributes to the pathogenesis of AIDS dementia complex. We report here on the effects of exogenous TNF-alpha on SK-N-MC human neuroblastoma cells differentiated to a neuronal phenotype with retinoic acid, TNF-alpha caused a dose-dependent loss of viability and a corresponding increase in apoptosis in differentiated SK-N-MC cells but not in undifferentiated cultures. Importantly, intracellular signalling via TNF receptors, as measured by activation of the transcription factor NF-kappa B, was unaltered by retinoic acid treatment. Finally, overexpression of bcl-2 or crmA conferred resistance to apoptosis mediated by TNF-alpha, as did the addition of the antioxidant N-acetylcysteine. These results suggest that TNF-alpha induces apoptosis in neuronal cells by a pathway that involves formation of reactive oxygen intermediates and which can be blocked by specific genetic interventions.

A transgenic mouse model for studying the lineage relationships and differentiation program of type II pneumocytes at various stages of lung development.

A pedigree of transgenic mice has been characterized that contains a H2-Kb/LacZ fusion gene that exhibits integration site-dependent expression from the earliest stages of lung development through adulthood. Histochemical and immunocytochemical studies indicate that the LacZ reporter appears throughout the pulmonary endoderm by embryonic day 11 (E11). A proximal-to-distal wave of extinction of transgene expression occurs during E13-14 that parallels the wave of cytodifferentiation of the pulmonary endoderm. By E16, the LacZ reporter is restricted to the distal portion of epithelial tubules and by birth to scattered cells located in alveoli. Crude epithelial cell suspensions were prepared from lungs harvested from E16 and 14 day postnatal transgenic mice, labeled with the fluorescent LacZ substrate fluorescein di-(beta-galactopyranoside), and the LacZ expressing population isolated by fluorescence-activated cell sorting. Electron microscopic, immunocytochemical and histochemical studies of this purified cell population establish that type II pneumocytes are the only cell lineage that support H2-Kb/LacZ expression in the mature postnatal lung. Fluorescence-activated cell sorting of E16 lung suspensions yielded a homogeneous population of cells that produced surfactant protein A, that could be maintained in cell culture, and that are likely precursors of adult type II pneumocytes. Together these studies indicate that (i) expression of the transgene in this pedigree of mice provides a marker for describing early differentiation of the pulmonary epithelium; (ii) the transgene may be useful as an enhancer trap to isolate cis-acting sequences that regulate gene transcription within this lineage; (iii) the LacZ reporter expression can be used to purify specific embryonic pulmonary epithelial cell populations; and (iv) primary cultures of these embryonic populations represent a potentially useful model system for analyzing the cellular components and signaling pathways necessary to support and complete passage through the type II pneumocyte differentiation program.

Integration site-dependent transgene expression used to mark subpopulations of cells in vivo: an example from the neuromuscular junction.

To produce transgenic mice, an exogenous gene is inserted into the germ line, usually by injection of the DNA construct into a pronucleus of a fertilized egg. In most cases the transgene is integrated into the genome at a single random site. Frequently, the transgenes are combinations of regulatory elements from one gene and protein coding sequences from another gene, the reporter. As expected, the promoter in the construct usually controls the expression pattern of the reporter. In some cases, however, transgenes have been constructed with regulatory elements that are not able to direct transcription on their own. In animals containing such transgenes, the expression of the reporter is dependent on endogenous regulatory elements near the chromosomal site of transgene integration. In the present study, an Escherichia coli beta-galactosidase (lacZ) reporter gene linked to a weak promoter was selectively expressed in discrete subpopulations of cells in each of eight independently derived lines of mice. In one line (line 42), which we analyzed in detail, a subset of cells in skeletal muscle were lacZ-positive. Specifically, fibroblasts close to neuromuscular junctions expressed the lacZ-protein, whereas skeletal muscle fibroblasts far from synaptic sites and fibroblasts in other organs were lacZ-negative. Moreover, Schwann cells at nerve terminals were lacZ-positive, whereas Schwann cells in extramuscular nerves were lacZ-negative. These results indicate the existence of differences between perisynaptic and extrasynaptic fibroblasts in normal skeletal muscle. They also demonstrate how such transgenic mice can be used to identify and mark discrete cell populations.

Integration site-dependent expression of a transgene reveals specialized features of cells associated with neuromuscular junctions.

After skeletal muscle is denervated, fibroblasts near neuromuscular junctions proliferate more than fibroblasts distant from synaptic sites, and they accumulate adhesive molecules such as tenascin (Gatchalian, C. L., M. Schachner, and J. R. Sanes. 1989. J. Cell Biol. 108:1873-1890). This response could reflect signals that arise perisynaptically after denervation, preexisting differences between perisynaptic and extrasynaptic fibroblasts, or both. Here, we describe a line of transgenic mice in which patterns of transgene expression provide direct evidence for differences between perisynaptic and extrasynaptic fibroblasts in normal muscle. Transgenic mice were generated using regulatory elements from a major histocompatibility complex (MHC) class I gene linked to the Escherichia coli beta-galactosidase (lacZ) gene. Expression of lacZ was detected histochemically. In each of eight lines, lacZ was detected in different subsets of cells, none of which included lymphocytes. In contrast, endogenous MHC is expressed in most tissues and at high levels in lymphocytes. Thus, the MHC gene sequences appeared inactive in the transgene, and lacZ expression was apparently controlled by genomic regulatory elements that were specific for the insertion site. In one line, cells close to the neuromuscular junction were lacZ positive in embryonic and young postnatal mice. Electron microscopy identified these cells as fibroblasts and Schwann cells associated with motor nerve terminals, as well as endoneurial fibroblasts, perineurial cells, and Schwann cells in the distal branches of motor nerves. No intramuscular cells greater than 200 microns from synaptic sites were lacZ positive. These results indicate that there are molecular differences between perisynaptic and extrasynaptic fibroblasts even in normal muscle and that diverse perisynaptic cell types share a specific pattern of gene expression.

Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.

Studies of interleukin 1 (IL-1) alpha and beta have emphasized their functional similarities. IL-1 alpha and -beta are encoded by ancestrally related genes that have diverged dramatically in primary sequence; however, only modest differences in the regulation or biological activity of IL-1 alpha and IL-1 beta have been documented. Here we show that mouse L cells transfected with murine pro-IL-1 alpha cDNA expressed biologically active, 33-kilodalton pro-IL-1 alpha, and that this pro molecule was neither processed to the 17-kilodalton mature form nor secreted. The transfected cells also expressed membrane-associated IL-1 biological activity, indicating that the pro-IL-1 alpha cDNA can direct expression of membrane-associated IL-1 and that cleavage of the pro molecule is not required for membrane presentation. In contrast, transfection of pro-IL-1 beta cDNA did not generate biologically active material in L cells. Evidence is presented that the native murine IL-1 beta precursor molecule is also biologically inactive in peritoneal exudate cells stimulated with lipopolysaccharide. These differences in distribution of the bioactive forms of IL-1 alpha and IL-1 beta may provide selective advantages for the maintenance of two gene products with similar functions.