RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

Cytokine signaling through the novel tyrosine kinase RAFTK in Kaposi's sarcoma cells.

A number of cytokines, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), oncostatin M (OSM), IL-6, and tumor necrosis factor alpha (TNF-alpha), have been postulated to have a role in the pathogenesis of Kaposi's sarcoma (KS). The proliferative effects of bFGF and OSM may be via their reported activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway in KS cells. We now report that KS cells express a recently identified focal adhesion kinase termed RAFTK which appears in other cell systems to coordinate surface signals between cytokine and integrin receptors and the cytoskeleton as well as act downstream to modulate JNK activation. We also report that the tyrosine kinase receptor FLT-4, present on normal lymphatic endothelium, is robustly expressed in KS cells. Treatment of KS cells with VEGF-related protein (VRP), the ligand for the FLT-4 receptor, as well as with the cytokines bFGF, OSM, IL-6, VEGF, or TNF-alpha resulted in phosphorylation and activation of RAFTK. Following its activation, there was an enhanced association of RAFTK with the cytoskeletal protein paxillin. This association was mediated by the hydrophobic COOH-terminal domain of the kinase. Furthermore, JNK activity was increased in KS cells after VEGF or VRP stimulation. We postulate that in these tumor cells RAFTK may be activated by a diverse group of stimulatory cytokines and facilitate signal transduction to the cytoskeleton and downstream to the growth promoting JNK pathway.

HIV-1 infection of human fetal thymocytes.

Some neonates with congenital human immunodeficiency virus type 1 (HIV-1) infection exhibit immune dysregulation. This suggests that fetal CD4+ cells, possibly thymocytes, may be infected during gestation. If thymocytes are infected, this may result in a disruption of T-cell differentiation. To examine this hypothesis, normal human fetal thymocytes were established in tissue culture, characterized, and then exposed to HIV-1. On initial isolation, fetal thymocytes were analyzed for phenotypic markers by flow cytometry and assessed for T-cell function by mitogen-stimulated thymidine incorporation. The thymocytes comprised greater than 70% double positive (CD4+, CD8+) cells and responded to T- but not to B-cell mitogens. Thereafter, thymocytes were incubated in either tissue culture medium containing infectious HIV-1 or in control (HIV-free) medium. Infection of fetal thymocytes was determined by light and electron microscopy in combination with immunocytochemistry, molecular hybridization, and an infectious cell center (ICC) assay. After 1 week in culture, the thymocytes exposed to HIV-1 were positive by immunocytochemistry for the HIV-1-associated protein gp41. In addition, the presence of HIV-1 DNA was detected by molecular hybridization confirming infection of these cells. The ICC assay demonstrated the production of infectious HIV-1 particles and budding of mature virions was observed by electron microscopy. These studies demonstrate that human fetal thymocytes can be infected with HIV-1 in vitro and that this infection results in production of infectious virions. These results support the hypothesis that vertical transmission of HIV-1 in vivo may result in the infection of fetal thymocytes, which may contribute to postnatal HIV-1-associated pathologic conditions.

Neural cell targets of human immunodeficiency virus type 1 in human fetal organotypic cultures.

Some children infected by HIV-1 demonstrate nervous system disease. Because a significant percentage of these children are believed to be infected during gestation and it is thought that HIV-1 may infect distinct glial populations, this work tested the hypothesis that different HIV-1 isolates can infect cells of the developing human fetal central nervous system (CNS). Central nervous system organotypic tissue cultures derived from human fetal brain enable the study of complex interactions between CNS cell types. Central nervous system organotypic cultures were exposed to lymphocytotropic (L-tropic) or monocytotropic (M-tropic) HIV-1 isolates and monitored for viral infection. HIV-1 gp41 and p24 antigens were detected by immunocytochemistry (ICC), HIV-1 RNA was localized in the cytoplasm of CNS cells by in situ hybridization (ISH), and viral DNA was detected by polymerase chain reaction (PCR) in HIV-1-exposed cultures. Double-label ICC identified HIV-1 antigens in both microglia and astrocytes. These results demonstrate that both L- and M-tropic isolates infect microglia and astrocytes in human fetal organotypic cultures. In addition, HIV-1 infection was detected in culture supernatants up to day 57 postinfection and at 90 days by coculture with susceptible CEM cells. HIV-1 infection of neural cells appears to be productive. This model may permit further examination of the interaction of HIV-1 with the developing human CNS and the mechanisms of AIDS-associated neuropathology.

Simple, rapid, quantitative, syncytium-forming microassay for the detection of human immunodeficiency virus neutralizing antibody.

A simple, rapid, quantitative syncytium-forming microassay for the detection of human immunodeficiency virus (HIV-I) isolates is described. A virus-syncytial sensitive clone of CEM cells (CEM-SS) was identified and made adherent to flat bottom 96-well microtiter dishes. Following the addition of virus, these cells develop easily quantifiable, adherent syncytia on a background of confluent, normal CEM-SS monolayer in 4 to 6 days. One-hit kinetics for syncytia formation were obtained at various multiplicities of infection. Syncytia are associated with complete virion production and cytoplasmic localization of the p24 core protein (detected by immunofluorescence). Total infectious virus can be accurately determined in this assay; these results showed a close correlation with p24 and gp120 induction when microtiter well supernatants were passed to fresh cells and evaluated by competitive radioimmunoassay. Studies of p24 antigen induction at and beyond the end point of syncytia formation indicate that there are no detectable nonsyncytial variants in standard HIV-I stocks. Six divergent HIV-I isolates (HTLV-IIIB, -RFII, -MN, -RUTZ, -CC, and LAV-1), as well as HTLV-IIIB and LAV-1 reisolated from persistently infected chimpanzees, produce quantifiable syncytia which vary slightly in their developmental morphology. Accurate neutralization titers are readily obtained from easily constructed multiplicity curves derived from serial dilutions of test sera. Inherent within this system is a flexible method for studying various kinetics of antibody/virus interactions, as well as blocking and interference studies with any candidate antiviral compounds.

Direct cytotoxicity of HIV-1 envelope protein gp120 on human NT neurons.

A new in vitro system comprising a pure population of neurons, human NT cells, was used to characterize the direct neurotoxic effect of HIV-1 envelope protein gp120. Cytotoxicity was monitored by a quantitative assay after exposure to recombinant gp120 in the presence or absence of other reagents. Treatment of mature NT neurons with various doses of gp120 for 24 h caused a decrease of up to 27% in the number of viable cells. This neurotoxicity was abolished by co-treatment with either D-2-amino-5-phosphonopentanoic acid (APV), MK801 or nimodipine, or by culturing cells in a Ca(2+)-free environment. Taken together, these data indicate that gp120 exerts a direct neurotoxic effect by acting through NMDA receptors and Ca2+ channels.

Human fetal myelinated organotypic cultures.

We have previously reported the establishment of organotypic cultures derived from human fetal brain tissue. Although these cultures permit the testing of multiple hypotheses about normal human neurodevelopment and neuropathologic conditions, they have the limitation of not being myelinated and therefore preclude the study of questions related to myelinogenesis and diseases of myelin. In the current communication, we describe recent developments that allow us to overcome this limitation and permit the establishment of a myelinated organotypic culture model. Sections of dorsal column dissected from the lumbar spinal cord of human fetuses ranging in age 21-23 weeks of gestation were placed in culture. The explants were maintained for up to 12 weeks during which time they were characterized and shown to express a number of CNS cell-type-specific markers including glial fibrillary acidic protein (astrocytes), nerve growth factor receptor and neurofilament protein (neurons), CD68 (microglia), and myelin basic protein, HNK-1 and galactocerebroside (oligodendrocytes). In addition, lectin histochemistry using Ricinus communis agglutinin-1 detected microglia and endothelial cells. Upon explantation, abundant myelin was seen by electron microscopy in the cultures. Although during the culture period there was degradation of myelin, there was also evidence of maintenance of intact myelin sheaths around small caliber axons and de novo myelin synthesis. This model system may permit the further use of human organotypic cultures to investigate issues related to neurodevelopment and to pathologic conditions including those relevant to dysmyelination and demyelination.

Human fetal central nervous system organotypic cultures.

Many aspects of human neurodevelopment remain enigmatic. A main reason for this is, although there have been a significant number of morphologic and biochemical studies of neural tissues derived from human embryos and fetuses, this can only provide a static picture of the events at a given gestational age. Also, in vitro studies that focus on cells derived from these tissues have a limitation in that different cell types in dissociated tissue culture cannot interact in a 'normal' physiologic manner thereby, perhaps, altering their housekeeping and luxury functions. The present study focused on the development of a human explant organotypic culture model that may overcome the static limitation of the first example and permit a dynamic analysis of different cell types as they interact which may satisfy the second restriction. Because there is an array of developmental markers that define different cell phenotypes, this explant model may also provide a means of analyzing, for the first time, processes undefined in the human CNS. Human fetal CNS tissue obtained from second trimester abortuses was established in culture. The tissues were maintained for up to 12 weeks during which time they developed and differentiated. Sample cultures were harvested periodically and analyzed by light- and electron microscopy in combination with immunocytochemistry. Differentiation of neurons, astrocytes, oligodendrocytes and endothelial cells was documented using morphologic and biochemical criteria. As such, this model system may allow for the analysis of processes that occur during normal human fetal neurodevelopment and in pathologic conditions.

The related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated and participates in colony-stimulating factor-1/macrophage colony-stimulating factor signaling in monocyte-macrophages.

RAFTK, a novel nonreceptor protein kinase, has been shown to be involved in focal adhesion signal transduction pathways in neuronal PC12 cells, megakaryocytes, platelets, and T cells. Because focal adhesions may modulate cytoskeletal functions and thereby alter phagocytosis, cell migration, and adhesion in monocyte-macrophages, we investigated the role of RAFTK signaling in these cells. RAFTK was abundantly expressed in THP1 monocytic cells as well as in primary alveolar and peripheral blood-derived macrophages. Colony-stimulating factor-1 (CSF-1)/macrophage colony-stimulating factor (M-CSF) stimulation of THP1 cells increased the tyrosine phosphorylation of RAFTK; similar increases in phosphorylation were also detected after lipopolysaccharide stimulation. RAFTK was phosphorylated with similar kinetics in THP1 cells and peripheral blood-derived macrophages. Immunoprecipitation analysis showed associations between RAFTK and the signaling molecule phosphatidylinositol-3 (PI-3) kinase. PI-3 kinase enzyme activity also coprecipitated with the RAFTK antibody, further confirming this association. The CSF-1/M-CSF receptor c-fms and RAFTK appeared to associate in response to CSF-1/M-CSF treatment of THP1 cells. Inhibition of RAFTK by a dominant-negative kinase mutant reduced CSF-1/M-CSF-induced MAPK activity. These data indicate that RAFTK participates in signal transduction pathways mediated by CSF-1/M-CSF, a cytokine that regulates monocyte-macrophage growth and function.

Beta-chemokine induction of activation protein-1 and cyclic AMP responsive element activation in human myeloid cells.

Chemokines effect leukocyte chemotaxis and activation through their binding to specific G protein-coupled receptors. Although early steps in chemokine signal transduction pathways have been characterized, there is relatively limited information available at the transcription factor level. To that end, we have examined the binding activity on activation protein-1 (AP-1) and cyclic AMP responsive element (CRE) target sequences in human THP-1 myeloid cells after treatment with the beta-chemokine macrophage inflammatory protein (MIP-1alpha). MIP-1alpha induced both AP-1 and CRE activation. Although inhibition of protein kinase C blocked the AP-1 activity induced by this chemokine, there was no decrease in CRE activation in the presence of a protein kinase A inhibitor. Using kinase assays, it appeared that mitogen-activated protein kinase pathways were involved in CRE activation. In addition, HIV-1 infection of THP-1 cells resulted in constitutive activation of AP-1 and CRE elements but no further response to MIP-1alpha treatment. These results suggest that beta-chemokines act via protein kinase C-dependent pathways and mitogen-activated protein kinase pathways to modulate the host transcriptional response in myeloid cells, and that this response is altered by HIV infection.

Productive infection of human fetal microglia by HIV-1.

Central nervous system disease is a frequent finding in both pediatric and adult AIDS. Microglia have been shown to be the major target of HIV-1 infection in the central nervous system. However, studies in vitro concerning susceptibility of human microglia to HIV-1 infection reported conflicting results; microglia from adult brain showed productive infection by HIV-1, whereas microglia from fetal brain did not. To investigate this further and to define the possible mechanisms responsible for this difference, we prepared highly purified human microglial cell cultures from fetuses of 16 to 24 weeks' gestation and exposed them to monocytotropic (HIV-1 JR-FL and HIV-1 JR-CSF) isolates of HIV-1. Culture supernatants were examined for the presence of p24 antigen for a 4-week period after viral exposure. Concurrently, potential cytopathic effects and cellular viral antigen expression (gp41 and p24) were examined by light microscopy in combination with immunocytochemistry. The results showed that human fetal microglia can be productively infected by HIV-1 as judged by p24 antigen capture assay, syncytia formation, and gp41 and p24 immunoreactivity of infected microglia. In addition, by electron microscopy, numerous viral particles characteristic of HIV-1 were present both in the intracellular and extracellular compartments. Uninfected cultures or astrocytes overgrown in the microglial cultures did not show evidence of infection under identical experimental conditions. These data demonstrate that human fetal microglia, like their adult counterparts, are susceptible to HIV-1 infection in vitro and can support the production of virus.

GM-CSF induces eosinophilic cell growth-promoting activity on human fetal liver cells.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been described as a multi-lineage growth factor that induces in vitro colony formation of bone marrow erythroid burst-forming units (BFU-E), multipotential colony-forming units (CFU-GEMM), granulocyte-macrophage CFU (CFU-GM), granulocyte CFU (CFU-G), macrophage CFU (CFU-M), as well as eosinophil colony-forming units (CFU-Eo). Because of the preeminent role of the liver in fetal hematopoiesis, the effect of human recombinant GM-CSF (hrGM-CSF) on hematopoietic cells isolated from human fetal liver was tested in liquid cultures and in semisolid colony assays. hrGM-CSF induced a significant increase in the number of mature eosinophils in liquid culture and to a lesser extent in semisolid cultures when compared to untreated culture controls. The kinetics of this effect on eosinophils reached its peak on day 21 of culture. When GM-CSF and erythropoietin (Ep) were added simultaneously to the cultures, no significant change in the number of eosinophils compared to hrGM-CSF alone was observed. Ep or granulocyte colony-stimulating factor (G-CSF) did not show any CFU-Eo activity when added separately or simultaneously to both liquid and semisolid cultures. These results indicate that hrGM-CSF alone may be a potent stimulating factor for CFU-Eo obtained from human fetal liver and, in combination with other growth factors, control optimal development of human fetal eosinophils.

Alterations in human fetal hematopoiesis are associated with maternal HIV infection.

In the majority of adult and pediatric patients with AIDS, hematologic abnormalities including leukopenia, anemia, and thrombocytopenia are commonly observed. In addition to these findings, changes in hematopoietic progenitor cells occur, including a reduction of multipotential-forming units, granulocyte-macrophages, macrophage as well as eosinophil colony-forming units, and bone marrow erythroid burst-forming units. This study examined alterations in human fetal liver hematopoiesis in 2nd trimester abortuses from human immunodeficiency virus (HIV)-seropositive women. The differentiation and growth potential of hematopoietic cells in vitro were monitored. Upon initial isolation, some populations of liver hematopoietic cells from abortuses of HIV-seropositive women were significantly decreased when compared to age-matched samples from fetuses of normal females including the percentage of early T cells [cluster of differentiation (CD)2], B cells (CD19), and early monocytes (CD14). A decrease in multipotent progenitors (CD34), myelomonocytes (CD33), and panleukocytes (CD45) was also observed. In contrast, after 21 d in culture, cells from HIV abortuses demonstrated an increase in the percentage of CD14 cells when stimulated with erythropoietin and granulocyte-monocyte colony-stimulating factor, as well as an increase in CD45 phenotype after exposure to granulocyte-monocyte colony-stimulating factor alone. These samples showed a persistence of erythropoietic elements (transferrin and CD36 phenotype) when compared to normal controls. No significant difference in the in vitro growth of hematopoietic progenitors (bone marrow erythroid burst-forming units, granulocyte-macrophage colony-forming units, and multipotential forming units) between these samples and normal controls was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent productive HIV-1 infection of a CD4- human fetal thymocyte line.

Human fetal thymuses were obtained from abortuses of HIV-1 seronegative females. Thymocytes were isolated and cultured for 2 days with PHA. Thereafter, the culture was divided and half of the cells were exposed to the HIV-1 RF isolate for 4 h. After this incubation period, the HIV-1 exposed and nonexposed control cells were cultured in RPMI 1640 supplemented with IL-2 for 30 days and subsequently maintained in RPMI without the addition of growth factors. Long term culture of both HIV-1 exposed and control thymocytes has yielded two cell lines that have been maintained for more than 3 yr without the addition of growth factors. Flow cytometry using mAb that recognize T cell differentiation markers was used to analyze cell phenotypes. The HIV-1 exposed thymocyte cell line (E88/RF) was shown to be HIV-1 infected by p24 ELISA, reverse transcriptase activity, immunocytochemistry, in situ hybridization, polymerase chain reaction, electron microscopy, and to produce infectious particles by a syncytial forming assay. The non-HIV-1-exposed thymocyte cell line (T412) has remained negative by all criteria for HIV-1 infection. Flow cytometry showed the T412 cells to be positive for the T cell markers CD45, CD38, and CD4 but negative for all other markers tested. The E88/RF cells are positive for CD45 and CD38 but negative for CD4 and other markers. These data report the isolation of two human fetal thymocyte cell lines; one uninfected and susceptible to HIV-1 infection, and the other persistently and productively infected with HIV-1 with little cytopathology. These findings suggest that HIV-1 can persistently infect early T cells and may alter T cell differentiation.

Differential effects of interleukin-13 on cytomegalovirus and human immunodeficiency virus infection in human alveolar macrophages.

Alveolar macrophages, which form a principal line of defense against a variety of pulmonary pathogens, may themselves be infected by viruses like human immunodeficiency virus-1 (HIV-1), which impair their defensive functions. Interleukin-13 (IL-13), a multifunctional cytokine, has been considered for therapeutic use based on its potent inhibition of HIV-1 in these cells. We have further examined the effects of IL-13 on alveolar macrophages under conditions that reflect those seen in acquired immune deficiency syndrome, where this cell type is often infected by the opportunistic pathogen human cytomegalovirus (HCMV). Alveolar macrophages exposed to both HCMV and HIV-1 consistently exhibited higher levels of HIV-1 replication than cells exposed to HIV-1 alone. HIV-1 production was strongly suppressed in alveolar macrophages treated with IL-13 regardless of whether or not the cultures were coinfected with HCMV. However, IL-13 treatment markedly enhanced the expression of HCMV in otherwise latently infected macrophages in a dose dependent manner. These unexpected differential effects of IL-13 on host-virus interactions are important considerations in guiding its potential therapeutic applications.

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.