Todd C. Pappas

Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding.

GH3/B6 rat pituitary tumor cells exhibit rapid prolactin release (within 5 min) when treated with nanomolar amounts of estrogen. However, the putative protein mediator of this nongenomic action has not been described. Using antibodies directed against a peptide representing the hinge region of the intracellular estrogen receptor (iER), we have demonstrated that these cells contain a membrane ER (mER). We now report that confocal scanning laser microscopy of cells labeled live with the anti‐peptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and a polyclonal antibody, ER21, each recognizing a unique epitope on iER (NH2 terminal to the DNA‐binding region, within the steroid binding region, and the NH2‐terminal end, respectively), also immunohistochemically label membrane proteins of immuno‐selected GH3/B6 cells. These cells also specifically bind a fluorescent estrogen‐BSA conjugate. Coincubation of cells with anti‐ER antibody and the fluorescent estrogen‐BSA conjugate reveals that these labels colocalize on cells. These results suggest that mER may be structurally similar to iER.—Pappas, T. C., Gametchu, B., Watson, C. S. Membrane estrogen receptors identified by multiple antibody labeling and impeded‐ligand binding. FASEB J. 9, 404–410 (1995)

Apoptosis of CD8 + T cells is mediated by macrophages through interaction of HIV gp120 with chemokine receptor CXCR4

CD8-positive T cells are thought to play an important role in the control of infection by human immunodeficiency virus (HIV) as a result of their cytotoxic activity and by releasing soluble factors,. In AIDS patients, the absolute number of CD8+ T lymphocytes is decreased in peripheral blood, and their turnover rate is increased, suggesting that there is more cell renewal and cell death occurring. Anti-retroviral therapy raises CD8+ T-cell counts in HIV-infected patients. Here we report that the death rate of CD8+ T cells by apoptosis increased markedly during HIV infection of peripheral blood mononuclear cells in vitro. Apoptosis is induced in a dose-dependent manner by recombinant envelope glycoprotein gp120 from HIV strain X4, or by stromal-derived factor-1 (SDF-1), the physiological ligand of the chemokine receptor CXCR4. Apoptosis is mediated by the interaction between tumour-necrosis factor-α bound to the membrane of macrophages (mbTNF) and a receptor on CD8+ T cells (TNF-receptor II, or TNFRII). The expression of both of these cell-surface proteins is upregulated by HIV infection or by treatment with recombinant gp120 or SDF-1. Apoptosis of CD8+ T cells isolated from HIV-infected patients is also mediated by macrophages through the interaction between mbTNF and TNFRII. These results indicate that the increased turnover of CD8+ T cells in HIV-infected subjects is mediated by the HIV envelope protein through the CXCR4 chemokine receptor.

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-α

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-alpha (mERalpha) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERalpha by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERalpha, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERalpha and elicits the same response. A total of eight antibodies to ERalpha recognize mERalpha, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERalpha on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERalpha-specific antibodies. The characteristics of mERalpha and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.

Composition of PLGA and PEI/DNA nanoparticles improves ultrasound-mediated gene delivery in solid tumors in vivo

The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

Potential Role for CD63 in CCR5-Mediated Human Immunodeficiency Virus Type 1 Infection of Macrophages

ABSTRACT Macrophages and CD4+ lymphocytes are the principal target cells for human immunodeficiency virus type 1 (HIV-1) infection, but the molecular details of infection may differ between these cell types. During studies to identify cellular molecules that could be involved in macrophage infection, we observed inhibition of HIV-1 infection of macrophages by monoclonal antibody (MAb) to the tetraspan transmembrane glycoprotein CD63. Pretreatment of primary macrophages with anti-CD63 MAb, but not MAbs to other macrophage cell surface tetraspanins (CD9, CD81, and CD82), was shown to inhibit infection by several R5 and dualtropic strains, but not by X4 isolates. The block to productive infection was postfusion, as assessed by macrophage cell-cell fusion assays, but was prior to reverse transcription, as determined by quantitative PCR assay for new viral DNA formation. The inhibitory effects of anti-CD63 in primary macrophages could not be explained by changes in the levels of CD4, CCR5, or β-chemokines. Infections of peripheral blood lymphocytes and certain cell lines were unaffected by treatment with anti-CD63, suggesting that the role of CD63 in HIV-1 infection may be specific for macrophages.

Membrane estrogen receptor-enriched GH3/B6 cells have an enhanced non-genomic response to estrogen

We immunoselected GH3/B6 cells for a membrane estrogen receptor (mER) using antibodies generated against the rat intracellular ER (iER). Immunocytochemistry with anti-ER antibodies revealed bright fluorescence distributed in patches over the surface of mER-enriched cells, while cells immuno-depleted for mER showed only low-level membrane immunofluorescence. Quantitation via digital image analysis confirmed that immunoenriched populations show increases in both stained cell numbers and intensity of staining. Short-term culturing with serum reversibly decreased the intensity of immunostaining in mER-enriched cells to immuno-depleted cell levels. The mER-enriched populations initially contained ∼85% immunopositive cells in defined medium, but when cultured continuously with serum gradually decline to ∼22% immunopositive cells by 10 weeks. Cells enriched for mER showed a significant increase in rapid (after 2 or 5 min) prolactin release when treated with 17β-estradiol, while mER-depleted cells lacked this response. Immunoprecipitabie membrane proteins isolated from mER-enriched cells were 60,000, 74,000 and ∼ 200,000 MW, compared to an iER size of 67,000. Therefore, the presence and level of an mER that is antigenically related to iER is correlated with the ability of GH3/B6 cells to mediate a rapid action of estrogen.

Dopamine neurotoxicity in cortical neurons

Dopamine (DA), at concentrations greater than 100 microM, has previously been demonstrated to be toxic to mesencephalic, striatal and dorsal root ganglion cell cultures. Pharmacological experiments suggest that DA also may play a role in the cortical neurotoxicity caused by systemic administration of N-methyl-D-aspartate receptor antagonists such as phencyclidine and MK-801. In this study, the potential toxicity of DA in primary cortical cell cultures was determined in vitro. Using calcein and ethidium homodimer fluorescence as a marker for live and dead cells, respectively, we observed that a 24 h treatment with 10-100 microM DA produced a concentration-dependent increase in the number of ethidium homodimer-labelled cells. The cell death induced by 10 microM DA was dramatically reduced by co-administration of either superoxide dismutase and catalase or deferoxamine mesylate, an iron chelator. To verify this observation, the effects of 10 microM DA on the release of cytoplasmic lactate dehydrogenase (LDH) was measured. DA increased LDH release in a manner that was inhibited by both superoxide dismutase/catalase and deferoxamine. Nomifensine potentiated the effect of DA on LDH release, suggesting a protective role for DA uptake in this system. On the other hand, neither D1 nor D2 antagonists were able to prevent DA-induced LDH release. These data suggest that relatively low concentrations of DA can be injurious to cortical neurons through a mechanism that likely involves DA autooxidation and the formation of reactive oxygen species such as superoxide anion and hydroxyl radical. This mechanism may be important in the toxic effects of psychomotor stimulants such as amphetamine. However, the failure of DA receptor antagonists to affect DA-induced injury argues that the effect of DA on cortical neurons in culture does not model the toxic effect of phencyclidine and MK-801 observed in vivo.

Cytokine/neurotrophin interaction in the aged central nervous system

Age‐associated neurodegenerative diseases such as Alzheimers disease are characterised by neuronal impairment that leads to cognitive deficits. As certain affected neurons depend on trophic factors such as neurotrophins (NTs), impairment in NT function has been suggested to be a component of neuronal damage associated with such disorders. Age‐related neurodegenerative diseases are also characterised by high levels of proinflammatory cytokines such as tumour necrosis factor alpha (TNFα) in the CNS. Because TNFα receptors and certain NT receptors share a high degree of homology and are capable of activating similar signalling pathways, one possibility is that altered cytokine levels may affect NT function in the aged or diseased CNS. Here we wish briefly to review the evidence suggesting a role for cytokine and NT in the onset of age‐associated neurodegenerative diseases. We propose that cytokine/NT interactions may alter neuronal homeostasis, thus possibly contributing to some of the neuronal degeneration occurring during such age‐associated CNS diseases.

Body-Image Distortion among Male and Female College and High School Students, and Eating-Disordered Patients

For 179 male and female college and high school students, and 26 female eating-disordered patients body-image distortion was measured using a computer-based image-analysis of redrawn images of standardized human figures. Statistical analysis indicated that body-image distortion was the same for all groups. Body-image distortion was significantly and negatively related to weight:heigbt ratio as a function of a simple polynomial. These results suggest this evaluation of distortion of body-image yields a quantitative measure reliably related to weight status but also suggests the technique, and possibly measurement of body-image distortion in general, may not be a valid discriminator between eating-disordered and normal persons.

Tumour necrosis factor‐alpha‐ vs. growth factor deprivation‐promoted cell death: different receptor requirements for mediating nerve growth factor‐promoted rescue

Physiological and pathological aging of the central nervous system (CNS) is characterized by functional neuronal impairments which may lead to perturbed cell homeostasis and eventually to neuronal death. Many toxic events may underlie age‐related neurodegeneration. These include the effects of beta amyloid, Tau and mutated presenilin proteins, free radicals and oxidative stress, pro‐inflammatory cytokines and lack of growth factor support, which can be individually or collectively involved. Taken individually, these toxicants can induce very diverse cell responses, thus requiring individually targeted corrective interventions upstream of common cell death (apoptotic) pathways. Recent preliminary evidence suggests that the pro‐inflammatory cytokine tumour necrosis factor alpha (TNFα) and growth factor withdrawal can both activate a common apoptotic pathway in nerve growth factor (NGF)‐responsive PC12 cells involving caspase 3, albeit through very distinct upstream pathways: the former through active signalling and the latter through passive or lack of survival signalling. Here, we show that NGF can rescue PC12 cells from both growth factor withdrawal‐ and TNFα‐promoted cell death. However, NGF rescue from growth factor withdrawal requires NGF signalling through the high‐affinity tyrosine kinase receptor (TrkA), while NGF rescue from TNFα‐promoted cell death requires NGF signalling through the low‐affinity p75NTR receptor. These results strengthen the idea that prevention of age‐ or pathology‐associated neurodegeneration may require varied molecular approaches reflecting the diversity of the toxicants involved, possibly acting simultaneously.