Matrix metalloproteinase-dependent shedding of intercellular adhesion molecule-5 occurs with long-term potentiation.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can be released or activated in a neuronal activity dependent manner. Although pathologically elevated levels of MMPs may be synaptotoxic, physiologically appropriate levels of MMPs may instead enhance synaptic transmission. MMP inhibitors can block long term potentiation (LTP), and at least one family member can affect an increase in the volume of dendritic spines. While the mechanism by which MMPs affect these changes is not completely understood, one possibility is that the cleavage of specific synaptic cell adhesion molecules plays a role. In the present study, we have examined the ability of neuronal activity to stimulate rapid MMP dependent shedding of the intercellular adhesion molecule-5 (ICAM-5), a synaptic adhesion molecule that is thought to inhibit the maturation and enlargement of dendritic spines. Since such cleavage would likely occur within minutes if it were relevant to a process such as LTP, we focused on post stimulus time points of 30 min or less. We show that NMDA can stimulate rapid shedding of ICAM-5 from cortical neurons in dissociated cell cultures and that such shedding is diminished by pretreatment of cultures with inhibitors that target MMP-3 and -9, proteases thought to influence synaptic plasticity. Additional studies suggest that MMP mediated cleavage of ICAM-5 occurs at amino acid 780, so that the major portion of the ectodomain is released. Since reductions in ICAM-5 have been linked to changes in dendritic spine morphology that are associated with LTP, we also examined the possibility that MMP dependent ICAM-5 shedding occurs following high frequency tetanic stimulation of murine hippocampal slices. Results show that the shedding of ICAM-5 occurs in association with LTP, and that both LTP and the associated ICAM-5 shedding are reduced when slices are pretreated with an MMP inhibitor. Together, these findings suggest that neuronal activity is linked to the shedding of a molecule that may inhibit dendritic spine enlargement and that MMPs can affect this change. While further studies will be necessary to determine the extent to which cleavage of ICAM-5 in particular contributes to MMP dependent LTP, our data support an emerging body of literature suggesting that MMPs are critical mediators of synaptic plasticity.

Matrix metalloproteinase-7 modulates synaptic vesicle recycling and induces atrophy of neuronal synapses.

Matrix metalloproteinase-7 (MMP-7) belongs to a family of zinc dependent endopeptidases that are expressed in a variety of tissues including the brain. MMPs are known to be potent mediators of pericellular proteolysis and likely mediators of dynamic remodelling of neuronal connections. While an association between proteases and the neuronal synapse is emerging, a full understanding of this relationship is lacking. Here, we show that MMP-7 alters the structure and function of presynaptic terminals without affecting neuronal survival. Bath application of recombinant MMP-7 to cultured rat neurons induced long-lasting inhibition of vesicular recycling as measured by synaptotagmin 1 antibody uptake assays and FM4-64 optical imaging. MMP-7 application resulted in reduced abundance of vesicular and active zone proteins locally within synaptic terminals although their general levels remained unaltered. Finally, chronic application of the protease resulted in synaptic atrophy, including smaller terminals and fewer synaptic vesicles, as determined by electron microscopy. Together these results suggest that MMP-7 is a potent modulator of synaptic vesicle recycling and synaptic ultrastructure and that elevated levels of the enzyme, as may occur with brain inflammation, may adversely influence neurotransmission.

The matrix metalloproteinases and CNS plasticity: an overview.

The matrix metalloproteinases (MMPs) are expressed in response to pro-inflammatory stimuli and other triggers. The MMPs cleave numerous substrates including extracellular matrix components, cytokines and growth factors. In the CNS, while most studied in the context of disease, the many physiological functions of the MMPs are now becoming appreciated. This review provides an overview of the growing body of evidence for physiological roles of MMPs both in CNS development and in CNS plasticity in normal brain functioning, including learning and memory, as well as in CNS repair and reorganization as part of the neuroimmune response to injury.

Glial activation and matrix metalloproteinase release in cerebral malaria.

Although neurological symptoms associated with cerebral malaria (CM) are largely reversible, recent studies suggest that lasting neurological sequelae can occur. This may be especially true for children, in whom persistent deficits include problems with memory and attention. Because the malaria parasite is not thought to enter the brain parenchyma, lasting deficits are likely related to factors including the host response to disease. Studies with a rodent model, and with human postmortem tissue, suggest that glial activation occurs with CM. In this review, the authors will highlight studies focused on such activation in CM. Likely causes will be discussed, which include ischemia and activation of blood brain barrier endothelial cells. The potential consequences of glial activation will also be discussed, highlighting the possibility that glial-derived proteinases contribute to structural damage of the central nervous system (CNS). Of note, for the purposes of this focused review, glial activation will refer to the activation of astrocytes and microglial cells; discussion of oligodendroglial cells will not be included. In addition, although events thought to be critical to the pathogenesis of CM and glial activation will be covered, a comprehensive review of cerebral malaria will not be presented. Excellent reviews are already available, including Coltel et al (2004; Curr Neurovasc Res 1: 91-110), Medana and Turner (2006; Int J Parasitol 36: 555-568), and Hunt et al (2006; Int J Parasitol 36: 569-582).

Interaction of HIV Tat and matrix metalloproteinase in HIV neuropathogenesis: a new host defense mechanism.

Tat, the HIV transactivating protein, and matrix metalloproteinases (MMPs), a family of extracellular matrix (ECM) endopeptidases, have been implicated in the pathogenesis of HIV-associated dementia. However, the possibility that MMPs interact with viral proteins has remained unexplored. We therefore treated mixed human fetal neuronal cultures with recombinant Tat and select MMPs. Neurotoxicity was determined by measuring mitochondrial membrane potential and neuronal cell death. Previous studies have shown that Tat and MMP independently cause neurotoxicity. Surprisingly, we found the combination of Tat and MMP produced significant attenuation of neurotoxicity. To determine whether there was a physical interaction between Tat and MMP, we used protein electrophoresis and Western blot techniques, and found that MMP-1 can degrade Tat. This effect was blocked by MMP inhibitors. Furthermore, MMP-1 decreased Tat-mediated transactivation of the HIV long terminal repeat region, and this functionality was restored when MMP-1 activity was inhibited. These results suggest that the decrease in Tat-induced neurotoxicity and HIV transactivation is due to Tat's enzymatic cleavage by MMP-1. The direct interaction of human MMPs with viral proteins has now been demonstrated, with resultant modulation of Tat-mediated neurotoxicity and transactivation. This study elucidates a unique viral-host interaction that may serve as an innate host defense mechanism.

Markers of immune activation and viral load in HIV-associated sensory neuropathy.

BACKGROUND: HIV infection is associated with a painful distal sensory polyneuropathy (DSP) that can severely limit the quality of life of affected subjects. The pathogenesis of DSP is unknown, although both HIV proteins and products of immune activation triggered by HIV infection have been implicated. OBJECTIVE: To assess the association between baseline markers of immune activation and HIV RNA levels (viral load) and time to symptomatic DSP (SDSP). METHODS: A cohort of 376 subjects, most receiving highly active antiretroviral therapy (HAART), were followed semiannually for up to 48 months. Blood and CSF levels of HIV viral load, monocyte chemotactic protein-1, macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase-2, and tumor necrosis factor-alpha were measured in addition to CD4 lymphocyte cell count. RESULTS: In subjects without SDSP at baseline (62.5% of the cohort), among the virologic and immunologic markers, only baseline CSF M-CSF levels were associated with time to SDSP (hazard ratio = 2.97, p = 0.05). The Kaplan-Meier estimate of the 1-year incidence of SDSP was 21%, a 15% decrease from that observed in the Dana cohort, a pre-HAART cohort enrolled with the same inclusion/exclusion criteria. CONCLUSION: Highly active retroviral therapy (HAART) has changed the natural history of HIV-associated symptomatic distal sensory polyneuropathy (SDSP), which may explain, in contrast with studies from the pre-HAART era, the lack of association between SDSP and baseline HIV viral load and CD4 cell count.

Evaluation of HIV RNA and markers of immune activation as predictors of HIV-associated dementia.

OBJECTIVE: To evaluate whether baseline levels of plasma and CSF HIV RNA, tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), or macrophage colony stimulating factor (M-CSF) are predictors of incident HIV-associated dementia (HIVD) in a cohort with advanced HIV infection. METHODS: A total of 203 nondemented subjects with CD4 lymphocyte counts less than 200/muL, or <300/microL but with cognitive impairment, underwent semiannual neurologic, cognitive, functional, and laboratory assessments. HIVD and minor cognitive motor disorder (MCMD) were defined using American Academy of Neurology criteria. The cumulative incidence of HIVD was estimated using Kaplan-Meier curves. Cox proportional hazards regression models were used to examine the associations between biologic variables and time to HIVD, adjusting for age, sex, years of education, duration of HIV infection, type of antiretroviral use, premorbid IQ score, and presence of MCMD. RESULTS: After a median follow-up time of 20.7 months, 74 (36%) subjects reached the HIVD endpoint. The dementia was mild in 70% of cases. The cumulative incidence of HIVD was 20% at 1 year and 33% at 2 years. Highly active antiretroviral therapy (HAART) was used by 73% of subjects at baseline. A plasma HIV RNA level was undetectable in 23% of subjects and a CSF HIV RNA level was undetectable in 48% of subjects. In adjusted analyses, neither plasma nor CSF HIV RNA levels (log10) were associated with time to HIVD; log10 levels of plasma TNFalpha (HR 3.07, p = 0.03) and CSF MCP-1 (HR = 3.36, p = 0.06) tended to be associated with time to HIVD. CONCLUSION: The lack of association between baseline plasma and CSF HIV RNA levels and incident dementia suggests highly active antiretroviral therapy may be affecting CNS viral dynamics, leading to lower HIV RNA levels, and therefore weakening the utility of baseline HIV RNA levels as predictors of HIV-associated dementia.

CSF soluble Fas correlates with the severity of HIV-associated dementia.

Soluble Fas (sFas) and soluble Fas ligand (sFasL) are associated with cellular dysfunction and death and are elevated in CSF from patients with HIV dementia (HIV-D). The authors investigated whether these markers correlated with dementia severity and course. sFas and sFasL were measured in 15 highly active antiretroviral therapy (HAART)-naïve HIV-D subjects, 30 HAART-naïve HIV+ controls, and 17 HIV-controls. HIV-D subjects had higher CSF sFas levels than controls. Subjects with moderate/severe dementia had higher CSF sFas levels than those with mild dementia. CSF sFas trended lower in those with progressive dementia.

Characterization of human cleaved N-CAM and association with schizophrenia.

The neural cell adhesion molecule (N-CAM) is a cell recognition molecule involved in cellular migration, synaptic plasticity, and CNS development. A 105- to 115-kDa isoform of N-CAM (cleaved N-CAM or cN-CAM) is increased in schizophrenia in hippocampus, prefrontal cortex, and CSF. We purified and partially characterized cN-CAM, a putative novel isoform, and confirmed that the first 9 amino acids were identical to exon 1 of N-CAM, without the signal sequence. Analysis of trypsin-digested cN-CAM fragments by matrix-assisted laser desorption ionization on a time-of-flight mass spectrometer (MALDI-TOF) yielded peptides that could be identified as being derived from the first 548 amino acid residues of the expected N-CAM amino acid sequence. Immunological identification with four specific N-CAM antisera directed toward cytoplasmic, secreted, variable alternative spliced exon, or GPI epitopes failed to indicate other known splice variants. Neuraminidase treatment of cN-CAM produced a minor alteration resulting in a faster migrating immunoreactive band, indicating partial glycosylation of cN-CAM. Membranous particles from cytosolic brain extract containing cN-CAM were obtained by ultracentrifugation; however, CSF contained few such particles. cN-CAM and synaptophysin were colocalized on these particles. Both cN-CAM and N-CAM 180 were present in synaptosomal preparations of human brain. Following incubation of synaptosomes or brain tissue without protease inhibitors, N-CAM 180 was degraded and cN-CAM was increased. A cN-CAM-like band was present in human fetal neuronal cultures, but not in fetal astrocyte cultures. Thus, cN-CAM represents a protease- and neuraminidase-susceptible fragment possibly derived by proteolytic cleavage of N-CAM 180. An enlargement in ventricular volume in a group of adult patients with schizophrenia over a 2-year interval was found to be correlated with CSF cN-CAM levels as measured at the time of the initial MRI scan (r = 0.53, P = 0.01). cN-CAM is associated with ventricular enlargement; thus, the release of N-CAM fragments may be part of the pathogenic mechanism of schizophrenia in vulnerable brain regions such as the hippocampus and prefrontal cortex. Alternatively, the increases in cN-CAM in schizophrenia may be a reflection of a more general abnormality in the regulation of proteolysis or of extracellular matrix stability.

HIV-1 Tat neurotoxicity is prevented by matrix metalloproteinase inhibitors.

The release of potentially neurotoxic molecules by HIV-infected brain macrophages is accompanied by neuronal injury and death that results in the development of HIV-associated dementia (HAD). Among the potential neurotoxins implicated in the development of HAD is the HIV-1 transactivating protein, Tat. To investigate the mechanism by which Tat causes neurotoxicity, brain-derived Tat sequences from nondemented (Tat-ND) and demented (Tat-HAD) AIDS patients, which differed primarily in the augmenting region of Tat, were expressed in U937 monoblastoid cells and primary human macrophages. Cells expressing Tat-HAD protein exhibited elevated matrix metalloproteinase (MMP)-2 and -7 release and activation, but cells expressing Tat-ND did not exhibit enhanced MMP expression. Conditioned media from Tat-HAD-transfected cells caused significantly greater neuronal death (15.4 +/- 4.3%) than did Tat-ND (4.4 +/- 2.1%) or nontransfected (2.1 +/- 0.8%) cell-derived conditioned media. The neurotoxicity induced by Tat-HAD was inhibited by anti-MMP-2 or -7 antibodies (p < 0.005) but not by antibodies against MMP-9 or Tat. Similarly, scid/nod mice receiving striatal implants of Tat-HAD-transfected cells exhibited greater neurobehavioral abnormalities and neuronal loss (p < 0.005) than did animals receiving Tat-ND or nontransfected cells, which were reduced by treatment with the MMP inhibitor prinomastat (p < 0.005). These findings indicate that Tat causes neuronal death through an indirect mechanism that is Tat sequence dependent and involves the induction of MMPs.

Matrix metalloprotease-9 release from monocytes increases as a function of differentiation: implications for neuroinflammation and neurodegeneration.

Naïve monocytes extravasate in response to monocyte chemoattractant-1 (MCP-1) and subsequently, following differentiation within tissue, carry out effector functions. Consistent with this concept, expression of the MCP-1 receptor CCR2 decreases with monocyte differentiation, as production of cytokines increases (Fantuzzi et al., 1999). Because matrix metalloproteases (MMPs) may also play an important role in the ability of monocytes to migrate into tissues and/or to promote pathogen clearance/tissue injury, we have examined production of matrix metalloprotease-9 as a function of both monocyte differentiation in vitro and expression of CCR2. Increased time in culture, which is linked to monocyte differentiation, resulted in enhanced production of MMP-9, assessed by gelatin substrate zymography. Further, CCR2-negative monocytes produced greater quantities of MMP-9 than did naïve CCR2-positive cells. Our results indicate that MMP-9 release increases during monocyte differentiation, consistent with a prominent role in effector functions. Because extracellular matrix proteins are important to cell structure and survival (Wee Yong et al., 1998), increased expression of MMP-9 could contribute to tissue damage following monocyte differentiation.

Cytotoxicity by matrix metalloprotease-1 in organotypic spinal cord and dissociated neuronal cultures.

Extracellular matrix (ECM) proteins, including collagens and laminins, are critical to the structure of the neuronal synapse and may also be involved in cell survival. In the present study, we therefore examined the possibility that select ECM degrading proteins might be toxic to organotypic spinal cord and dissociated neuronal cultures. Of those proteins tested, including MMP-1, -7, and -9, we observed that MMP-1 was toxic to spinal cord cultures as determined by release of lactic acid dehydrogenase as well as uptake of propidium iodide. Pretreatment of cell cultures with 50 microM alpha-tocopherol partially reversed these effects. We also observed that MMP-1 was toxic to human neurons grown in dissociated cultures and that increased amounts of MMP-1 were released by astrocytes following their stimulation with IL-1beta. These results suggest that further studies may be warranted to determine whether MMP-1 contributes to neurodegenerative conditions in which activated astrocytes may play a role.

The role of chemokines in human immunodeficiency virus infection.

Chemokines that bind to human immunodeficiency virus (HIV) co-receptors are potent and selective inhibitors of HIV infection. Therefore, ever since our discovery of this activity, we have proposed their role in controlling HIV infection as a third arm of the immune response, i.e. in concert with humoral and cellular responses. Research carried out in our laboratory, and performed independently by other groups, has recently strengthened this concept. Here, we critically analyze the evidence indicating the positive contribution of chemokines to HIV infection, their involvement with cognate and innate immunity, and the potential for their use in combating HIV infection.

Cerebrospinal fluid levels of MMP-2, 7, and 9 are elevated in association with human immunodeficiency virus dementia.

Pathological evidence suggests that alterations of the blood-brain barrier (BBB) may occur in association with human immunodeficiency virus (HIV) dementia (HIVD). Increased BBB permeability could contribute to the development of dementia by facilitating the entry of activated and infected monocytes, as well as potentially toxic serum proteins, into the central nervous system. One mechanism by which BBB permeability may be altered is through increased activity of select matrix metalloproteinases (MMPs). In the present study, we examined the possibility that MMPs that target critical BBB proteins, including laminin, entactin, and collagen type IV, are elevated in the cerebrospinal fluid (CSF) of patients with HIVD. We also examined the possibility that such MMPs could be produced by brain-derived cells, and that MMP production by these cells might be increased by tumor necrosis factor-alpha, an inflammatory cytokine that is produced by HIV-infected monocytes/microglia and is elevated in HIVD. By using western blot and enzyme-linked immunosorbent assay, we observed that CSF levels of pro-MMP-2 and pro-MMP-7 were increased in association with HIVD. In addition, through the use of gelatin substrate zymography, a sensitive functional assay for MMP-2 and MMP-9, we observed that MMP-2 or pro-MMP-9 activity was more frequently detectable in the CSF of individuals with HIV dementia (9/16) than in the CSF from either nondemented seropositive (2/11) or seronegative (0/11) controls. Although the presence of MMPs in the serum could contribute to elevated levels in the CSF, we also show that brain-derived cells release MMP-2, 7, and 9, and that such release is increased after their stimulation with tumor necrosis factor-alpha. Together, these results suggest that elevated CSF levels of select MMPs may reflect immune activation within the central nervous system. They also suggest that further studies may be warranted to determine whether these proteins may play a role in the development of symptomatic neurological disease.

Transient exposure to HIV-1 Tat protein results in cytokine production in macrophages and astrocytes. A hit and run phenomenon.

The pathological correlates of dementia due to human immunodeficiency virus (HIV) infection are glial cell activation and cytokine dysregulation. These findings occur in the setting of small numbers of productively infected cells within the brain. We determined whether exposure of susceptible cells to Tat protein of HIV could result in the production of select proinflammatory cytokines. In a dose-responsive manner, Tat induced interleukin (IL)-1beta production in monocytic cells, while astrocytic cells showed an increase in mRNA for IL-1beta, but had a translation block for IL-1beta protein production. Conversely, IL-6 protein and mRNA productions were strongly induced in astrocytic cells and minimally in monocytic cells. IL-1beta and IL-6 production were independent of tumor necrosis factor-alpha production. An exposure to Tat for a few minutes was sufficient for sustained releases of cytokines for several hours. This prolonged cytokine production is likely maintained by a positive feed back loop of Tat-induced nuclear factor kappaB activation and cytokine production that is independent of extracellular calcium. Thus a transient exposure may be sufficient to initiate a cascade of events resulting in cerebral dysfunction and a "hit and run" approach may be in effect. Hence cross-sectional measurement of viral load in the brain may not be a useful indicator of the role of viral products in the neuropathogenesis of HIV dementia.

IFN-gamma inhibits AP-1 binding activity in human brain-derived cells through a nitric oxide dependent mechanism.

It has been demonstrated that CNS levels of the cytokine IFN-gamma are elevated in association with a number of neuro-inflammatory diseases. In the present study, we have examined the effect of this cytokine on human brain derived cells. We show that prolonged treatment (22 h) of such cells with IFN-gamma inhibits the DNA binding activity of transcription factor AP-1. Furthermore, we show that this effect can be reversed by either N(G)-monomethyl-L-arginine (L-NMMA) or L-N5-(1-iminoethyl)ornithine (L-NIO), competitive inhibitors of nitric oxide synthase activity [Rees et al., 1990]. In addition, we show that treatment of brain-derived cells with the nitric oxide donor 3-morpholinosydnonimine, HCl (SIN-1), or [N-(b-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide] (glyco-SNAP-1), also inhibits the binding activity of AP-1. Together, these results suggest that IFN-gamma can inhibit AP-1 binding activity through a nitric oxide dependent mechanism.

Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia.

Activated monocytes release a number of substances, including inflammatory cytokines and eicosanoids, that are highly toxic to cells of the central nervous system. Because monocytic infiltration of the central nervous system closely correlates with HIV-1-associated dementia, it has been suggested that monocyte-derived toxins mediate nervous system damage. In the present study, we show that the HIV-1 transactivator protein Tat significantly increases astrocytic expression and release of monocyte chemoattractant protein-1 (MCP-1). Astrocytic release of beta-chemokines, which are relatively less selective for monocytes, including RANTES, macrophage inflammatory protein-1alpha, and macrophage inflammatory protein-1beta, was not observed. We also show that MCP-1 is expressed in the brains of patients with HIV-1-associated dementia and that, of the beta-chemokines tested, only MCP-1 could be detected in the cerebrospinal fluid of patients with this condition. Together, these data provide a potential link between the presence of HIV-1 in the brain and the monocytic infiltration that may substantially contribute to dementia.

Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection has been associated with an increase in the binding of the transcription factor NF-kappa B to its consensus sequence in the viral promoter. Using cultures of primary human fetal astrocytes, we show that exogenous HIV-1 Tat protein, which has been demonstrated to be released from infected cells, is associated with an increase in the binding of this transcription factor to an HIV-1 long terminal repeat kappa B sequence. This effect occurs rapidly and is independent of new protein synthesis. We also demonstrate that extracellular Tat protein is associated with an increase in protein kinase C activity. If Tat functions similarly in other cell types, such findings could relate to some of this protein's previously described physiological effects. These effects include Tat's ability to upregulate the synthesis of specific cytokines and to act as a growth factor.

Laminar distribution of MK-801, kainate, AMPA, and muscimol binding sites in cat visual cortex: a developmental study.

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the sensitive period for monocular deprivation in the visual cortex. To study glutamate receptors, we incubated sections of cat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e-maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [3H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [3H]kainate and [3H]AMPA are competitive ligands for non-NMDA receptors. We used [3H]muscimol, which binds to GABAA receptors, so that we would have one control ligand that binds to a nonglutamate receptor. When all layers were combined, the results confirmed our previous studies with homogenate binding. [3H]MK-801 and [3H]kainate binding were significantly greater at 42 days than at earlier or later times. [3H]AMPA and [3H]muscimol binding did not show such a peak. This suggests that MK-801 and kainate binding sites are more likely to be involved in plasticity than are AMPA and muscimol binding sites. In layers 2/3, MK-801 had the greatest age-dependent changes; in layers 5 and 6, kainate binding changed most with age. This suggests that the mechanisms of plasticity may vary with cortical layer.

An increase in p50/p65 NF-kB binding to the HIV-1 LTR is not sufficient to increase viral expression in the primary human astrocyte.

Human astrocytes can be infected with HIV-1 both in vivo and in vitro. The amount of HIV-1 p24 structural protein production is low in comparison to that of the macrophage. Several weeks following infection or transfection, however, cocultivation with uninfected lymphocytes or stimulation with the cytokines TNF-alpha and IL 1-beta will increase viral production from this cell type. In the present study we demonstrate that phorbol 12-myristate 13-acetate (PMA) also increases HIV-1 p24 production from the primary human astrocyte. Using electrophoretic mobility shift assay (EMSA) in combination with supershift studies using specific antibodies, we demonstrate that PMA, like TNF-alpha, increases the p50/p65 form of NF-kB. Furthermore we demonstrate that the protein kinase inhibitor H7 inhibits PMA- and TNF-alpha-associated increases in HIV-1 expression at a time when it has little to no inhibitory effect on the associated increases in p50/p65 NF-kB. Thus, unless p50/p65 NF-kB or its binding is affected by H7 in a manner that cannot be resolved by EMSA, an increase in this form of NF-kB is not always sufficient to increase HIV-1 expression from the astrocyte.