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Biomedical subjects

A Takashima

Publications and source records attributed to A Takashima.

At least 145 records · Page 8Linked to original sources
PubMed1993

Cloning and characterization of the 2B4 gene encoding a molecule associated with non-MHC-restricted killing mediated by activated natural killer cells and T cells.

We have recently described a signal transducing molecule, 2B4, expressed on all NK and T cells that mediate non-MHC-restricted killing. The gene encoding this molecule was cloned and its nucleotide sequence determined. The encoded protein of 398 amino acids has a leader peptide of 18 amino acids and a transmembrane region of 24 amino acids. The predicted protein has eight N-linked glycosylation sites, suggesting that it is highly glycosylated. Comparison of 2B4 with sequences in the databanks indicates that 2B4 is a member of Ig supergene family, and it shows homology to murine and rat CD48 and human LFA-3. Northern blot analysis has shown at least three transcripts for 2B4 in adherent lymphokine-activated killer cells of several mouse strains and TCR-gamma/delta dendritic epidermal T cell lines but not in allospecific T cell clones. These three mRNA are the products of differential splicing of heterogeneous nuclear RNA. Southern blot analysis of genomic DNA from several mouse strains revealed that 2B4 belongs to a family of closely related genes. The 2B4 gene has been mapped to mouse chromosome 1 by analysis of 2B4 expression in recombinant inbred mouse strains.

Amino Acid Sequence↗
PubMed1993

Killing of skin-derived tumor cells by mouse dendritic epidermal T-cells.

Dendritic epidermal T-cells (DETC) are a unique population of T-cells that reside normally in mouse epidermis and express a gamma delta T-cell receptor. We have reported previously that DETC acquire in culture the capacity to lyse the YAC-1 lymphoma, a conventional target for natural killer cells. The aim of the present study was to characterize this cytotoxic potential, using a spectrum of skin-derived mouse tumors. Cytotoxicity was measured by a 51Cr release assay and by the visual assessment of target cell lysis. Long-term DETC lines, established from CBA, AKR, and BALB/c mice by mitogenic stimulation and repeated feeding with interleukin 2 (5 units/ml), were used as effectors. Skin-derived tumor targets included 5 melanoma lines and the transformed keratinocyte line Pam 212. Each DETC line lysed skin-derived tumors as well as YAC-1 targets effectively in the 18-h 51Cr release assay, and target lysis occurred in a non-major histocompatibility complex-restricted manner. By contrast, freshly isolated spleen cells lysed YAC-1 but not skin tumor targets. Moreover, confluent monolayers of melanoma or Pam 212 targets were disrupted completely by added DETC lines but not by spleen cells. The cytolytic activity of DETC appeared to be specific for tumor cells, since normal mouse keratinocyte monolayers remained intact under the same conditions. Finally, DETC freshly isolated from skin failed to exhibit significant cytotoxicity but acquired this capacity 10-14 days after mitogenic stimulation and feeding with interleukin 2 (5 units/ml). We conclude that DETC possess the potential to recognize, bind, and lyse tumor cells that originate in skin.

Animals↗
PubMed1993

Tau protein kinase I is essential for amyloid beta-protein-induced neurotoxicity.

Pathological changes of Alzheimer disease are characterized by cerebral cortical atrophy as a result of degeneration and loss of neurons. Typical histological lesions include numerous senile plaques composed of deposits of amyloid beta-protein and neurofibrillary tangles consisting predominantly of ubiquitin and highly phosphorylated tau proteins. Previously, tau protein kinase I (TPK I) was purified and its cDNA was cloned. To examine the biological role of this enzyme in neurons, we have studied the induction of its kinase activity in primary cultures of embryonic rat hippocampal neurons. Treatment of cultures with amyloid beta-protein significantly increased TPK I activity and induced the appearance of tau proteins recognized by the Alz-50 monoclonal antibody. In addition, though amyloid beta-protein was neurotoxic, either cycloheximide or actinomycin D prevented neuronal death. Death was also prevented by TPK I antisense oligonucleotides but not by sense oligonucleotides. These observations suggest that rat hippocampal neurons undergo programmed cell death in response to amyloid beta-protein and that TPK I is a key enzyme in this process.

Amyloid beta-Peptides↗
PubMed1993

Regulation of muscarinic stimulation of norepinephrine release and PI hydrolysis during cell growth in PC12 cells.

We have studied the relationships of cell growth to muscarinic stimulation of norepinephrine release and phosphoinositide hydrolysis in the rat pheochromocytoma PC12 cells. The ability of these cells to release norepinephrine in response to muscarinic agonists was maximal during the early phase of exponential growth, and then rapidly decreased to undetectable levels as the cells approached stationary phase. In contrast, muscarinic stimulation of phosphoinositide hydrolysis was low in the early exponential phase of growth, increased to a maximum during late exponential growth and then dramatically dropped in the stationary phase. The number of muscarinic receptors, as measured by antagonist-binding studies, also varied during cell growth with maximal levels at days 2 and 8, corresponding to the maxima in muscarinic-stimulated norepinephrine release and phosphoinositide hydrolysis, respectively.

Animals↗
PubMed1993

In vivo activation of mouse dendritic epidermal T cells in sites of contact dermatitis.

Adult mouse epidermis contains a population of dendritic, Thy-1+, CD3+, CD4-, CD8- and T cell receptor (TcR) V gamma 3/V delta 1+ leukocytes termed dendritic epidermal T cells (DETC). DETC isolated from skin and placed into culture will proliferative vigorously in response to T cell mitogens and T cell growth factors. In the present study, we examined whether DETC can be activated in situ by modulating their epidermal environment. Ear skin of CBA mice was painted with the chemical irritant, croton oil, and the epidermal cells (EC) isolated from such sites were then tested for proliferative responses to exogenous interleukin-2 (IL-2), in the absence of added mitogens. Cells from croton oil-treated skin showed marked IL-2 responsiveness, whereas cells from phosphate-buffered saline-treated skin failed to proliferate. IL-2 responses were seen as early as 2 days after croton oil treatment and peaked between days 5 and 10. gamma delta TcR-bearing cells, most likely resident DETC, were the major population to respond to IL-2, since depletion of gamma delta TcR+ cells, but not alpha beta TcR+ cells, abolished that responsiveness, and since gamma delta TcR+ cell numbers increased markedly in the cultures that contained added IL-2. These results indicate that DETC in normal skin, which are at a state of rest, may be activated when their residential epidermal environment is disrupted externally. This process of DETC activation may be a critical step in the maturation of DETC into effector leukocytes in vivo.

Animals↗
PubMed1993

Signal transduction via phosphorylated adhesion molecule, LFA-1 beta (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells.

It is well known that IL-2 stimulates natural killer (NK) cells to express lymphokine activated killer (LAK) activity and that this stimulation prompts the acquisition of the ability to lyse previously insensitive target cells. The possible role of adhesion molecules in the IL-2 activation process was probed by focussing on a lymphocyte function-associated antigen (LFA)-1-dependent model system. A mAb to the LFA-1 beta chain abrogated LAK activity, but only moderately suppressed NK activity, suggesting a differential role for LFA-1 beta in LAK compared with NK mediated lysis. Orthophosphate labeling demonstrated that the LFA-1 beta chain was strongly phosphorylated in LAK but not NK cells; in contrast, the alpha chain was phosphorylated similarly in both effector cell types. At least a portion of the phosphorylation of the beta chain was on tyrosine residues, as shown by Western blotting with anti-phosphotyrosine antibody of LFA-1 beta immunoprecipitates. Crosslinking of the LFA-1 beta chain with plastic-adhered antibody stimulated Ca(2+)-dependent release of cytoplasmic lytic granules and induced phosphatidyl inositol turnover in LAK but not NK cells. We conclude that the IL-2-induced phosphorylation of the beta chain of the LFA-1 adhesion molecule in LAK cells and associated alteration in signal transduction may be important in the stimulation of LAK cell activity in NK cells.

Antigens, CD↗
PubMed1993

Dendritic epidermal T cells: lessons from mice for humans.

Dendritic epidermal T cells (DETC) in mice form part of a primitive system of epithelial-resident T cells characterized by the expression of gamma delta T-cell receptors (TCR). Critical attributes that characterize DETC include their highly restricted T-cell receptor gene utilization, proliferation and maturation within epidermis, a capacity to kill relevant skin-derived tumor targets, and the ability to modulate immune responses that are initiated and expressed in skin. Contemporary knowledge suggests that DETC and the related skin-directed gamma delta T cells found in humans play important roles in maintaining the immunologic integrity of skin.

Animals↗
PubMed1993

Mouse dendritic epidermal T cells exhibit chemotactic migration toward PAM 212 keratinocyte culture supernatants.

Dendritic epidermal T cells (DETCs) are Thy-1+, CD45+, CD3+, CD4-, CD8-, and T-cell receptor-V gamma 3/V delta 1+ leukocytes that reside normally in adult mouse skin. We have demonstrated previously that keratinocytes serve as adhesion substrates for DETCs, and that interleukin 7 (IL-7), which is produced by keratinocytes, serves as a growth factor for DETCs. The present study was conducted to address the mechanisms by which DETCs migrate into the epidermis, reasoning that keratinocytes may also be a source of chemotactic activity. Short-term DETC lines were 35S-labeled and tested for migration toward Pam 212 keratinocyte culture supernatants using a modified Boyden chamber method; cell movement from upper chambers toward test samples in lower chambers was traced by counting radioactivity. DETC displayed rapid (within 60 min) and marked (> 50%) migration toward keratinocyte supernatants. The majority of cells that had migrated into keratinocyte supernatants expressed the V gamma 3 T-cell receptor, thus verifying that the migrating cells were DETCs. Addition of keratinocyte supernatants to the upper chambers completely blocked migration, suggesting its chemotactic nature. By contrast, no DETC migration was observed toward 3T3 fibroblast supernatants. Chemotactic activities were 1) produced by Pam 212 cells even in the absence of serum; 2) greater than 12 kD in size; 3) heat and pH labile; 4) trypsin sensitive; and 5) precipitated by 60-100% ammonium sulfate. Several cytokines (e.g., IL-1 alpha and IL-8) failed to mediate DETC migration when added to the lower chambers. Likewise, the same cytokines, when added to the upper chambers, failed to inhibit DETC migration toward Pam 212 supernatants. These results support our hypothesis that keratinocytes facilitate the residence of DETC in epidermis by secreting unique chemotactic factors, by providing adhesion substrates, and by elaborating specific growth factors.

Animals↗
PubMed1993

Profiles of cytokine mRNA expressed by dendritic epidermal T cells in mice.

The epidermis of mice contains, in addition to Langerhans cells, a second dendritic population that is Thy-1+/CD3+/CD4-/CD8-/T-cell receptor-V gamma 3/V delta 1+. These dendritic epidermal T cells (DETC) are now thought to comprise one element in the family of epithelial tissue-resident gamma delta T cells. In the present study, DETCs were examined for their expression of mRNA for cytokines, using a reverse transcription-polymerase chain reaction. Freshly isolated Thy-1+ epidermal cells constitutively expressed mRNA for gamma-interferon, but not IL-2. Within 24 h after stimulation with Con A, these cells then expressed mRNA for gamma-interferon and IL-2, but not IL-4. The rapid onset of expression of mRNA for IL-2 occurred exclusively within the Thy-1+ population, and in a Con A-dependent fashion. When freshly isolated epidermal cells were first stimulated with Con A and then expanded in bulk with rIL-2 for 20-24 d, cells expressing IL-4 mRNA then emerged, upon secondary stimulation with Con A. These "short-term" DETC lines also expressed mRNA for IL-2, interferon-gamma, IL-1 alpha, IL-3, IL-6, IL-7, tumor necrosis factor alpha and beta, and granulocyte macrophage-colony stimulating factor. Interestingly, mRNA for IL-4 and IL-6 was no longer detected in long-term (> 1 year) DETC lines 7-17 and 12-12. In addition, one line (7-17) maintained IL-3 mRNA expression, whereas another (12-12) had lost this capacity. These results emphasize the concept that, as resident cells in epidermis, DETCs exhibit several different immunorelevant activities, and the heterogeneity in cytokine mRNA profiles suggests that DETCs may divide into functional subsets.

Animals↗
PubMed1993

Reciprocal cytokine-mediated cellular interactions in mouse epidermis: promotion of gamma delta T-cell growth by IL-7 and TNF alpha and inhibition of keratinocyte growth by gamma IFN.

A unique subset of gamma delta T cells, termed dendritic epidermal T cells (DETC), resides in symbiosis with keratinocytes in mouse epidermis. We have shown previously that interleukin 7 (IL-7) which is produced by keratinocytes, promotes growth and prevents apoptosis in DETC. To extend this observation, we examined 12 cytokines, each of which is expressed by epidermal cells at mRNA and/or protein levels, for their capacities to modulate the growth of DETC. Cytokines examined included IL-1 alpha, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), granulocyte/macrophage-colony stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha). When tested individually, IL-2 and IL-7 promoted maximal growth of the long-term cultured DETC line 7-17. When tested in combinations, synergistic growth-promoting effects were seen with IL-2 and IL-4 or IL-7, and with IL-7 and IL-4 or TNF alpha. Dose-response experiments demonstrated that TNF alpha, which is produced by keratinocytes, enhances IL-7-induced DETC proliferation, but inhibits IL-2-induced proliferation. The mouse keratinocyte-derived cell line Pam 212 was used to test these cytokines for their capacities to regulate keratinocyte growth. Only gamma IFN, which is produced by DETC, inhibited proliferation in a dose-dependent fashion. These results illustrate three reciprocal pathways by which epidermal cytokines regulate the growth of epidermal cells: 1) a paracrine mechanism by which keratinocyte-derived cytokines (e.g., IL-7 and TNF alpha) promote the growth of DETC, 2) an autocrine mechanism by which DETC-derived cytokines (e.g., IL-2 and IL-4) support their own growth, and 3) a reciprocal pathway in which a cytokine produced by resident epidermal leukocytes (e.g., gamma IFN) modulates the growth of keratinocytes.

Animals↗
PubMed1992

Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate.

The folate receptor mediates the uptake of 5-methyltetrahydrofolate in certain cultured cells by a process called potocytosis. When these cells are grown in physiological concentrations of folate, the receptor increases the efficiency of vitamin uptake by 30-fold. We now show that PAM 212 cells, a mouse keratinocyte cell line, are unable to grow in 1 nM 5-methyltetrahydrofolate unless they express a functional folate receptor. These results suggest that under certain conditions, tissue cells in the body may depend on the folate receptor to obtain enough 5-methyltetrahydrofolate for growth.

Animals↗
PubMed1992

Determination of the action spectrum for UV-induced plasminogen activator synthesis in mouse keratinocytes in vitro.

Mouse epidermal keratinocyte-derived Pam 212 cells were irradiated with UV light, and the culture media were examined for plasminogen activator (PA) activity by measuring the capacity to convert exogenous plasminogen into plasmin. Exposure of cells to a broad spectrum of light in the UVB range induced a significant elevation of PA activity at 16 h after irradiation. A dose-response study revealed that a maximal enhancement, 15-fold higher than non-irradiated controls, was induced at a sublethal UVB dose of 100 J/m2, which significantly inhibited cell proliferation without affecting cell viability. Addition of 5 micrograms/ml of cycloheximide lowered the UV-induced elevation of PA activity, suggesting that protein synthesis is required for this phenomenon. Action spectra for PA synthesis were obtained by irradiating cells with monochromatic light ranging from 250 to 360 mm, and the data demonstrated that the action spectrum was 250-320 nm in length with a peak between 260 and 280 nm. The results suggest that UV exposure is an important physiological trigger for modulating PA synthesis in the epidermis.

Animals↗
PubMed1992

Langerhans cells are the major source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated mouse epidermal cells.

Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.

Animals↗
PubMed1992

Cytokine expression by epidermal cell subpopulations.

Epidermal cells (EC) are a rich source of cytokines that can regulate the function of cells in skin and in other tissues. To organize the array of data pertaining to cytokine expression by EC subpopulations, we have tabulated such data according to cell source, state of cell activation, and type of assay employed. This information forms a background for our own studies, in which reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show that Langerhans cells (LC) are the principal source of mRNA for interleukin 1 beta and macrophage inflammatory protein-1 alpha (MIP-1 alpha) among unstimulated mouse EC.

Animals↗