EP0555231A1 - Regulation de la proliferation de cellules neoplasiques par l'intermediaire d'un nouveau recepteur 5ht1a - Google Patents

Regulation de la proliferation de cellules neoplasiques par l'intermediaire d'un nouveau recepteur 5ht1a

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Publication number
EP0555231A1
EP0555231A1 EP91915814A EP91915814A EP0555231A1 EP 0555231 A1 EP0555231 A1 EP 0555231A1 EP 91915814 A EP91915814 A EP 91915814A EP 91915814 A EP91915814 A EP 91915814A EP 0555231 A1 EP0555231 A1 EP 0555231A1
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Prior art keywords
cells
receptor
proliferation
cell
binding
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German (de)
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Thomas Martin Aune
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Bayer Corp
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Miles Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/286—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against neuromediator receptors, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the characterization of 5HT specific receptors on Jurkat cells and activated T cells by a combination of pharmacological, biochemical and molecular analyses has confirmed the presence of 5HT receptors and studies link the receptor on Jurkat cells and activated T cell to the 5HTla family of receptors. For Jurkat cells and activated T cells this is based on the following criteria: binding studies with the 5HTla receptor specific agonist (80HDPAT), studies on signal transduction, northern analysis with specific human 5HTla oligionucleotide probes. Resting T cells did not express 5HTla receptors by these same criteria.
  • the present invention relates to regulation of cell proliferation based on the recognition of a novel serotonin receptor present as a cell surface molecule.
  • the novel receptor is a serotonin receptor linked to the 5HT2 family.
  • the 5HT2-like receptor is present on neoplastic or tumor cells and activated T-cells, e.g. CD4+ and CD8+.
  • the present invention also relates to the regulation of cell proliferation based on the recognition that proliferating cells contain/express serotonin.
  • the proliferation of neoplastic or tumor cells and activated T-cells containing serotonin can be decreased by inhibition of serotonin synthesis.
  • the present invention also relates to the regulation of the proliferation of cell exhibiting the novel 5HT2 5 receptor by introducing an effective amount of agonists or antagonists to either increase or decrease cell proliferation.
  • serotonin (5-hydroxytryptamine, 5-HT)
  • 5-HT 5-hydroxytryptamine
  • Serotonin has been shown to augment IFN induced phagocytosis (5) and suppress IFN induced 5 la expression on macrophages (6), to augment NK cell cytotoxicity (7), to affect ion permeability in isolated pre-B lymphocytes (8), and to suppress mitogen stimulated T cell proliferation in in vitro systems (9).
  • serotonin and serotonin receptors have been suggested to be required for expression of delayed type hypersensitivity in murine models (10-11).
  • SUBSTITUTE SHEET 5HT antagonists have suggested that T lymphocytes which can transfer delayed type hypersensitivity responses to naive recipients may express 5HT receptors (11) • Specific 5HT2 antagonists such as ketanserin or c ritanserin will prevent transfer of this response.
  • 5HT receptor subtypes could be identified on human T lymphocytes and if 5HT receptors could mediate signal transduction events similar to those previously reported 0 for 5HT receptors in the nervous system.
  • the results show that a human T lymphocyte line (Jurkat) expresses a 5HT2 like-receptor subtype which can mediate phoshatidylinositol hydrolysis and intracellular Ca++ mobilization.
  • T lymphocytes The activation of T lymphocytes is a consequence of ligand-receptor interactions that occur at the interface of the T cell and an antigen-presenting cell.
  • a number of different cell surface molecules on the T lymphocyte and the antigen-presenting cell may
  • antigen-induced T lymphocyte activation must involve stimulation of the T cell antigen receptor. Stimulation of the T cell antigen receptor alone is insufficient to induce proliferative
  • T cells 30 responses.
  • Other cell surface molecules expressed on T cells function as accessory molecules.
  • Accessory molecules may function as adhesion molecules, modify the transmembrane signal initiated via the antigen receptor and/or initiate their own transmembrane signaling events.
  • T cell surface molecules appear on the surface of the T cell during the events associated with the activation, differentiation, and proliferation of T
  • T cell proliferation is believed to be regulated primarily through the actions of IL-2 on its specific cell surface receptor.
  • the role of IL-2 includes both autocrine and paracrine effects resulting in the proliferation of other T cells.
  • IL-2 driven T cell proliferation in considered the major mechanism responsible for T cell growth, under some circumstances T cell proliferation occurs independent of IL-2.
  • T cell proliferation occurs independent of IL-2.
  • the novel 5HT2-like receptor is present as a cell surface molecule on activated T cells, it has been determined that the novel 5HT2-like can be regulated by inhibiting serotonin synthesis.
  • the present invention also relates to the regulation of the proliferation of cell exhibiting the novel 5HT2 antagonists to either increase or decrease cell proliferation.
  • the present invention also relates to regulation of cell proliferation exhibiting the novel 5HT2-like receptor.
  • Said antibodies include a plurality of "types" of antibodies having an epitope or epitopes specific for the 5HT2-like receptor.
  • Such antibody “types” may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized or human antibodies.
  • the present invention also relates to the regulation of cell proliferation exhibiting the novel 5HT2—like receptor by generating imotopes, small peptide ligands, that bind to either the 5HT2-like receptor or to antibodies to the 5HT2-like receptor.
  • small peptide ligands would function as agonists and/or antagonists.
  • Proliferation of normal diploid cells generally requires the presence of a continuous supply of endogenous or exogenous growth factors. Proliferation of tumor cells has generally been found not to require addition of exogenous growth factors. This is due to the ability of tumor cells to produce their own growth
  • SUBSTITUTE SHEET factors due to changes in growth factor receptors so they are continuously activated or changes in signal transduction elements which result in the continuous activation of tumor cells.
  • Tumor cells may also express unique receptors or produce unique hormones or growth factors which are not usually associated with the parent tissue. Recently it has been shown that expression of 5HTlc or 5HT2 receptors (by gene transfeetion) in normal diploid murine fibroblasts results in the acquisition of
  • transfected cells express high affinity receptors for 5HT which transduce second messenger signals. These fibroblasts also acquired the ability to form foci in tissue culture,
  • a method of down-regulating proliferation of cells exhibiting a 5HT2-like receptor comprising, functionally decreasing the amount of serotonin available for binding to said 5HT2-like receptor or functionally reducing the availability of 5HT2-like receptor binding sites.
  • the 3 method applies to neoplastic or tumor cells and activated T-cells.
  • the activated T-cells can be IL-2 dependent or IL-2 independent.
  • the proliferation of the cells is
  • SUBSTITUTE SHEET decreased by administering a sufficient amount of a compound to inhibit the activity of the enzyme tryptophan hydroxylase thereby inhibiting serotonin synthesis.
  • An example of such a compound is p-chlorophenylalanine.
  • 5 A method of down-regulating proliferation of cells exhibiting a 5HT2-like receptor comprising functionally reducing the availability of 5HT2-like receptor binding sites, the proliferation is decreased by introducing an amount of at least one antagonist sufficient to bind to 0 said 5HT2-like receptor to interrupt cell proliferation.
  • Said at least one antagonist is selected from the class of 5HT receptor ligands.
  • Said class of 5HT receptor ligands includes ritanserin, mesulergine, mianserin, spiperone, mimotopes and antibodies to said 5HT2-like -.I- receptor.
  • a method of up-regulating proliferation of cells exhibiting a 5HT2-like receptor comprising functionally increasing the availability of 5HT2-like receptor binding sites.
  • the proliferation of said cells is increased by 20 introducing an amount of at least one agonist sufficient to enhance cell proliferation.
  • Said at least one agonist is selected from the class of 5HT receptor ligands.
  • Said class of 5HT receptor ligands include pelanserin, ketanserin, methyl serotonin, 8-OH-DPAT, and propranolol.
  • a method of treating a T-cell dependent disease state in a mammal comprising down-regulating proliferation of cells exhibiting a 5HT2-like receptor by functionally decreasing the amount of serotonin available for binding to said 5HT2-like receptor or functionally reducing the 30 availability of 5HT2-like receptor binding sites.
  • the proliferation of the cells is decreased by administering an effective amount of a compound to inhibit the activity of the enzyme tryptophan hydroxylase thereby inhibiting serotonin synthesis.
  • a compound includes 35 p-chlorophenylalanine.
  • a method of treating T-cell dependent disease state in a mammal comprising down-regulating the proliferation of cells exhibiting a 5HT2-like receptor by functionally reducing the availability of 5HT2-like receptor binding sites wherein the proliferation of said cells is decreased by introducing an effective amount of at least one antagonist sufficient to bind to said 5HT2-like receptor to interrupt cell proliferation.
  • Said at least one antagonist is selected from the class of 5HT receptor ligands.
  • Said class of 5HT receptor ligands include ritanserin, mesulergine, pirenperone, spiperone, mimotopes and antibodies to said 5HT2-like receptor.
  • a method of treating a neoplastic disease state in a mammal comprising down-regulating proliferation of cells exhibiting a 5HT2-like receptor by functionally decreasing the amount of serotonin available for binding to said 5HT2-like receptor or functionally reducing the availability of 5HT2-like receptor binding sites.
  • the proliferation of the cells is decreased by administering an effective amount of a compound to inhibit the activity of the enzyme tryptophan hydroxylase thereby inhibiting serotonin synthesis.
  • Such compounds include p-chlorophenylalanine.
  • a method of treating a neoplastic disease state in a mammal comprising down-regulating proliferation of cells exhibiting a 5HT2-like receptor by functionally reducing the availability of 5HT2-like receptor binding sites wherein the proliferation of said cells is decreased by introducing an effective amount of at least one antagonist sufficient to bind to said 5HT2-like receptor to interrupt cell proliferation.
  • Said at least one antagonist is selected from the class of 5HT receptor ligands.
  • Said class of 5HT receptor ligands include ritanserin, mesulergine, pirenperone, spiperone, mimotopes and antibodies to said 5HT2-like receptors.
  • a method of treating an immune deficient disease state in a mammal by up-regulating proliferation of T-cells exhibiting a 5HT2-like receptor comprising functionally increasing the availability of 5HT2-like receptor binding sites.
  • the proliferation of said cells is increased by introducing an effective amount of at least one agonist sufficient to enhance cell proliferation.
  • Said at least one agonist is selected from the class of 5HT receptor ligands.
  • Said class of 5HT receptor ligands include pelanserin, ketanserin, methyl serotonin, 8-OH-DPAT, propranolol, and mianserin.
  • Figure 1 is a graph demonstrating 5HT binding to Jurkat cells.
  • Jurkat cells (1 x 10 ) were incubated with the indicated concentrations of ( 3H)5HT for 6 min at 4oC in the absence (total binding ) or presence of 50 ⁇ M 5HT
  • the inset shows the results graphed on a Scratchard plot.
  • Figure 2 is a graph demonstrating kinetics of association
  • t- Figure 3 is a graph demonstrating comparison of the effect of OKT3 and 5HT on intracellular Ca 2+ concentrations in Jurkat cells. Approximately 5 s after data collection was initiated. IKT3 (1 ⁇ g/ml) or 5HT
  • Figure 4 is a graph demonstrating concentration dependence of 5HT mediated increases in intracellular
  • Figure 5 is a graph demonstrating 5HT-mediated increase in phosphatidylinositol turnover.
  • Figure 6A is a graph showing specific binding of 3H-5HT to T-cell blasts.
  • Figure 6B is a graph showing binding of ketanserin to T-cell blasts.
  • Figure 7 is a graph showing competition experiments with various 5HT receptor ligands.
  • Figure 8 is a graph showing the identification of 5HT in PBL.
  • Figure 9 is a graph showing effects of 5HT on proliferation of PBL.
  • Figure 10 is a graph showing analogues which bind to 5HT receptors and increase or decrease T-cell proliferation.
  • Figure 11 is a graph showing the activation of CD8+ suppressor T-cells by PWM requires 5HT.
  • Figure 12 is a graph showing the effects PBL stimulated with OKT3 in presence or absence of analogues which bind to 5HT.
  • FIGS 13A and 13B are graphs of properties of the serotonin receptor on tumor cells.
  • (A) Jurkat cells were harvested from log phase growth and incubated at 3x10 /ml in a total volume of 600 ⁇ l of RPMI 1640 media at 0-4°C in the presence of 3H-5HT ( ⁇ ), 1251-LSD ( ) , 3H-ketanserin ( ⁇ ) , 3H-80H-DPAT (0) or 3H-DOB(£7) (NEN) in the presence or absence of 50 ⁇ M 5HT for 6 min. Binding equilibrium was reached during this period (not shown) . Cells were collected on to nitrocellulose membranes (Millipore) and tubes and filters were washed three times each with 4ml of cold PBS. Ascorbate
  • the inset to BA shows a Scatchard analysis of binding data with 5HT ( ⁇ ) and ketanserin ( ⁇ ) .
  • the Kd was 130 nM.
  • Figure 14 is a graph showing reversal of ritanserin mediated inhibition of tumor cell proliferation by 5HT receptor ligands.
  • ME180 cells were cultured for 48 hr in the presence or absence of ritanserin (20 ⁇ M) and in the presence or absence of the indicated concentrations of the following 5HT receptor ligands: 5HT (CD), pelanserin ( ⁇ ) i Ketanserin ( ⁇ f ⁇ ⁇ ) , mianserin ( ) , 80H-DPAT( ⁇ ⁇ ) , propranolol ( ⁇ ) .
  • Cells were labeled with 1 ⁇ Ci 3H-TdR for the final 6 hr of culture and collected to measure incorporation into DNA. Results are expressed as the following 5HT receptor ligands: 5HT (CD), pelanserin ( ⁇ ) i Ketanserin ( ⁇ f ⁇ ⁇ ) , mianserin ( ) , 80H-DPAT( ⁇
  • Figure 15 is a graph showing 5HT dependent changes in intracellular cAMP content of cell blasts.
  • Figure 16 is a graph showing the rate of change of cAMP content and proliferation in T cell cultures after addition of 5HT.
  • Figure 17 is a graph showing inhibition of antiproliferative effects on gamma interferon (IFN).
  • ME-180 cells were cultured for three days with IFN in the absence ( ⁇ ) or presence of 300 ⁇ M tryptophan (M ⁇ ) or
  • Figure 18 is a graph showing a comparison of the ability of 5Htp and metabolites of 5Htp to reverse antiproliferative effects of gamma IFN.
  • ME-180 cells were cultured with 100 u/ml IFN in the presence or absence of the indicated concentrations of 5Htp (Q) , 5HT ( ⁇ ) , melatonin ( ⁇ ⁇ ) , N-methyl serotonin ( D ) or 5-hydroxyindole acetic acid (X) for three days.
  • Control proliferation was 73,456 + 2289 cpm and proliferation in the presence of IFN was 16,894 + 1145.
  • EET Figure 19 is a graph showing cells loss tryptophan and 5HT after culture with gamma IFN.
  • ME-180 cells were cultured for the indicated number of days in the presence (# / #) or absence (0,0) of 100 u/ml IFN , harvested and analyzed for 5HT content and tryptophan content by HPLC; LEFT GRAPH: 5HT; RIGHT GRAPH: tryptophan.
  • Figure 20 is a graph showing 5Htp does not inhibit loss of extracellular tryptophan in cultures with gamma IFN.
  • ME-180 cells were cultured in the absence (0) or presence of 100 u/ml IFN (#,D) and in the presence ( ⁇ ) or absence (O) of 10 ⁇ M 5Htp. Culture media was harvested on the indicated days and analyzed for tryptophan content by HPLC.
  • Figure 21 is a graph showing 5Htp reverses inhibition of cell proliferation by low tryptophan.
  • ME-180 cells were cultured for 48 hr in RPMI-1640 media with the indicated amounts of tryptophan with (#) or without (0) 3 ⁇ M 5Htp.
  • IP inositol phosphates
  • IC50 concentration required to inhibit response by 50%
  • EC50 concentration required to yield half-maximal response.
  • Jurkat cells were obtained from the American Type Culture Collection
  • EDTA incubated for 10 min on ice, and were homogenized with a Dounce homogenizer in 20-40 ml total volume. Homogenates were centrifuged at 10,000xg for 10 min to remove nuclei and cellular debris. The supernatant was centrifuged at 100,000xg for 1 hr. The pellet was suspended in tris-HCl buffer with EDTA at a final concentration of 7.7 mg/ml protein. This membrane preparation was stored at -70°C until use in binding assays.
  • Binding assays were performed in duplicate a minimum of three times.
  • DMEM with 10 mM HEPES for 45 min. at 37°C washed three 5 times and resuspended at 10 cells/ml in PBS. Aliquots (1 ml) were equilibrated to 37°C and placed in the modified sample station and data acquisition was initiated.
  • OKT3 (see text for concentrations) was added directly to 0 the sample and data acquisition continued for a total of
  • Fluorescence emission was divided by a longpass filter (455 nm cutoff) placed 45° to the collecting lens and measured simultaneously through two bandpass filters, BP485/22 and BP395/20. Data collected
  • Kd is the dissociation constant (250 nm)
  • R, Rmin, and Rmax are the fluorescence intensity ratios at resting, zero, and saturation calcium concentrations, respectively
  • Sf2/Sb2 is the ratio of fluorescence intensity of the calcium-free and calcium-bound dye (18).
  • PBMC Purification of PBMC.
  • PBMC were obtained from the buffy coats of whole blood from normal human volunteers.
  • PWM and PHA were obtained from GIBCO (0KT3 was from ascites of BALB/c mice and used at a dilution of 1:1000), diluted in PBS and used at a dilution of 1/80 in culture.
  • Purified recombinant or natural IL-2 were purchased from Boehringer Mannheim (Indianapolis, IN). One unit of IL-2 activity is defined as the amount required to cause 50% maximal proliferation of the IL-2 dependent cell line CTLL in a 24-h assay.
  • Purified rIL-1, rIL-4, and rIL-6 were obtained from Genzyme (Boston, MA) .
  • ME-180 cervical carcinoma cells were obtained from ATCC and were maintained as described ( ) . Cells were cultured for 48 hrs and cultures were either analyzed for 5HT content or
  • the retention times of known standards were as follows: 5HT, 3.50 min; 5OH-tryptophan, 4.77 min; tryptophan, 9.11 min, 50H-indoleacetic acid, 12.5 min; and melatonin 21.69 min.
  • Cells were also pulsed with 1 ⁇ Ci 3H-TdR for the final 4 hrs of culture and collected to measure incorporation into DNA. Cultures were performed in triplicate. Actual synthesis of 5HT from tryptophan was also demonstrated by culturing cells with 3-H-tryptophan, extracting soluble components and analyzing 3H-5HT by thin layer chromatography with a solvent system of methyl propanol-ethyl acetate-ammonium hydroxide-water
  • T-cells may express specific binding sites for this neurotransmitter. These cells have been used by numerous investigators to explore signal transduction mechanisms following antigen or mitogen stimulation (20-21).
  • Binding of 5HT reached a maximum at 1-2 ⁇ M 5HT.
  • the inset to figure 1 shows the Scatchard plot of the binding data. Binding data from three separate experiments yielded an average dissociation constant (Kd) of 90 nM.
  • 5HT up to 10 ⁇ M did not alter observed binding constants or receptor densities (data not shown) . All results shown here were derived from log phase cultures. Jurkat cells from cultures exceeding 10 cells/ml had significantly fewer 5HT receptors. These cultures also failed to respond to 5HT by mobilizing Ca++ or increasing inositol phosphate concentrations. Isolated membrane preparations also bound 5HT with similar characteristics. ' Binding of 5HT reached saturation at 1-2 ⁇ M 5HT and the average Kd determined by Scatchard analysis was 160 nM. Kinetics of binding and dissociation of 5HT (see below) were also similar for intact cells and membrane preparations. Similar concentrations of 5HT or 5HT agonists or antagonists
  • the receptor-ligand complex readily dissociated in the presence of excess unlabeled 5HT.
  • 5-HTlc or 5-HT2 sites results in increase in inositol phosphate levels and intracellular Ca++ concentration.
  • Jurkat cells were incubated with indo-1, a fluorescent dye sensitive to changes in intracellular Ca++, and analyzed by flow cytometry as described in Methods after incubation in the presence or absence of 5 ⁇ M 5-HT or 1 ⁇ g/ml OKT3. These results are shown in figure 3.
  • Cells incubated in PBS had a baseline calcium concentration of 150-175 nM and did not change intracellular Ca++ during the 10 min. observation period.
  • Jurkat cells incubated with OKT3 increased intracellular Ca++ to a maximum of 2 ⁇ M within 2 min. before decreasing to a level of 700 nM.
  • Jurkat cells incubated with 5 ⁇ M 5HT increased Ca++ concentration to 400 nM within 2 min.
  • FIG. 4 compares the increase in intracellular Ca++ concentration in cultures of Jurkat cells to Ca++ concentration in individual Jurkat cells as a function of 5HT concentration. At the indicated 5HT concentrations cells could be divided into those with low Ca++ (150-175 nM) and those with high Ca++ (375-425 nM) . Thus the response of the entire culture shown in figure 3 is actually the average of individual cells which either have high or low intracellular Ca++ concentrations. Increases in intracellular Ca++ concentration or the number of cells with increased intracellular Ca++. The half-maximal response was between 100-300 nM 5-HT which was approximately equivalent to the dissociation constant obtained from the binding data.
  • IP3 which is produced by hydrolysis of phosphatidylinositol biophosphate (22). Binding of 5-HT to the 5HT2 receptor family has been shown to increase levels of inositol phosphates in neural tissue.
  • 3 T-cells were labeled for 48 hrs. with H-inositol and stimulated with 3 ⁇ M 5-HT or with 1 ⁇ g/ml OKT3.
  • Cells were harvested at various times after addition of 5HT or OKT3 and analyzed for levels of IP by anion exchange chromatography. Results in fig. 5 show that IP levels increased after addition of 5HT or OKT3 and reached a maximum within 1 min. Levels of IP decreased over the next ten minutes approaching baseline levels. Both 5HT and OKT3 yielded similar increases in IP levels under these conditions.
  • 5-HT agonists or antagonists have been employed to help discriminate among the different 5-HT receptor subtypes.
  • Ketanserin, a 5-HT2 receptor antagonist, alpha-methyl serotonin, a 5HT2 receptor 3 agonist, and 5HT inhibited binding of H-5HT to Jurkat cells.
  • Concentrations which inhibited 50% of specific binding of H-5HT (IC50) were 20 ⁇ M, 3 ⁇ M and 0.8 ⁇ M for ketanserin, alpha-methyl serotonin and 5HT, respectively.
  • ICS-205930 a specific antagonist of the 5HT3 receptor failed to prevent 3H-5HT binding to Jurkat cells.
  • Other 5HT2 receptor antagonists such as ritanserin and pelanserin also failed to inhibit 5HT binding (not shown) .
  • H-ketanserin and H-DOB have been used to label 5HTlc or 5HT2 sites in the central nervous system.
  • 3 H-80HDPAT has been used to label 5HTla sites in the central nervous system.
  • Table 2 compares binding of these ligands to Jurkat cells as reflected by the Kd determined by Scatchard analysis. Specific binding was determined in the presence of lOO ⁇ M 5HT.
  • 5HT receptor agonists and antagonists have affinity constants in the range of 1-5 nM for their specific receptor classes.
  • the 5HT2 agonist, alpha methyl serotonin, was also tested for its ability to stimulate Ca++ mobilization
  • Jurkat cells (1x10 /ml) were incubated for 5 min. in the presence of 10 ⁇ M of the indicated antagonists before addition of 5HT.
  • Ca++ mobilization in Jurkat cells was determined by flow cytometry as described in the methods section 2 min. after addition of 5HT. At this time the response to 5HT is still linear with respect to time (Fig. 4).
  • IC50 is the concentration of agonist which inhibits 50% of Ca++ mobilization by Jurkat cells in the presence of 1 ⁇ M 5HT.
  • the neurotransmitter, 5HT is released during inflammation and has been suggested to play an important role in delayed type hypersensitivity responses (3,4,10).
  • Antagonists of 5HT such as ketanserin or ritanserin, have been shown to prevent DTH responses in animal models (10-11).
  • treatment of certain antigen specific T-cell clones with 5HT antagonists blocks their ability to transfer DTH responses to naive recipients.
  • functional 5HT receptors which transduce intracellular signals through second messengers have not been defined on T-cells. The purpose of experiments described here was to determine by both biochemical and pharmacological analysis whether functional receptors for 5HT could be identified on human T-cells and whether they were similar to subtypes already defined on other cell types present in the central orperipheral nervous system.
  • Receptor subtypes for 5HT have been characterized functionally, pharmacologically and molecularly (12-13).
  • the 5HT1 family consists of 5HTla, 5HTlb, and 5HTld
  • the 5HT2 family consists of 5HTlc, 5HT2a and 5HT2b
  • the 5HT3 family presently consists of only one member.
  • cDNA's encoding 5HTla, 5HTlc and 5HT2 receptors have been identified and all encode proteins which are G protein-linked receptors with seven membrane spanning units (23-25). Functionally, binding of 5HT to 5HTla sites results in modulation of adenylate cyclase activity (26), alterations in potassium ion channels, and inhibition of nerve cell transmissions (12-13).
  • Specific agonists have also been identified which exclusively bind to 5HTla sites.
  • 5HTlc and 5HT2 sites with equivalent affinities and examples include mesulergine and mianserin.
  • Other 5HT2 antagonists include ketanserin, ritanserin, and pelanserin. These ligands have been used to label 5HT2 sites, posses nanomolar affinities for 5HT2 sites and inhibit 5HT-mediated phosphatidyl-inositol turnover in target tissues (12-13).
  • the 5HT2 family may contain additional heterogeneity. 5HT sites which mediate phosphatidyl inositol turnover have also been identified in the hippocampus region of the brain as well as in the limbic forebrain.
  • 5 receptors appear to be localized primarily in the peripheral nervous system. These receptors are not affected by 5HT1 and/or 5HT2 selective ligands. However, specific ligands, such as ICS 205903, have been identified which can block the effects of 5HT in the 0 periphery (12-13).
  • the Kd for 5HT binding to Jurkat cells was 90 nM when determined by Scatchard analysis and was 180 nM when determined by kinetic analysis.
  • the site density for 5HT receptors was 130 fmol per million Jurkat cells or 80,000
  • 5HT stimulated phosphatidylinositol turnover and increases in intracellular Ca++ in these cells. Saturation of 5HT binding as well as maximum Ca++ response was observed at 1-3 ⁇ M 5HT. Half-maximal Ca++ responses were seen at 200 nM 5HT which is close to the
  • 5HT3 ligand ICS-205930 did not modulate cAMP concentrations in Jurkat cells (data not).
  • 125 I-LSD displaced by 5HT
  • mianserin, mesulergine, pelanserin, and ritanserin did not inhibit 5HT binding to Jurkat cells at concentrations 20 ⁇ M and did
  • 5HT receptors on human Jurkat T-cells and show that the receptor stimulates phosphatidyl-inositol turnover and increases in intracellular Ca++ concentration in these cells.
  • the increase in levels of inositol phosphate caused by 5HT was similar to the increase caused by OKT3.
  • the increase in Ca++ concentration by 5HT was from 175 nM to 400 nM within 2 min.
  • the increase in Ca++ concentration caused by OKT3 was to a maximum of 2 ⁇ M within 2 min. This increase rapidly decreased to 700 nM within an additional min.
  • 5HT phosphatidylinositol turnover represents a critical element of signal transduction in T-cell activation (for review see ref. 29) and stimulation of this pathway by 5HT should have important immunological consequences.
  • Sufficient 5HT may exist at sites of inflammation to modulate T-cell function.
  • 5HT is a major storage product of platelets and is released upon platelet aggregation which occurs at sites of inflammation (2-4).
  • 5HT levels in platelets are depressed in humans with autoimmune disease such as rheumatoid arthritis and systemic lupus erythematosus (30).
  • 5HT2 antagonists have been shown to exacerbate inflammatory and arthritic responses to streptococcal cell wall extracts in normally resistant rats (31).
  • 5HT receptors are mediated through 5HT receptors on T cells.
  • 5HT antagonists or agonists it should be possible to define the immunological consequences of interaction between 5HT and 5HT receptors on activated T cells and better understand the role of 5HT receptors on T cells in immune and inflammatory responses iji vivo.
  • similar types of 5HT receptors are present on human peripheral T cell blasts but are absent on resting human peripheral T cells (in preparation).
  • 5HT receptors on activated T cells The 5HT receptor on Jurkat cells has a weak affinity for H-5HT (100 nM) and for ketanserin (100 nM) when compared to classic 5HT2 receptors. Specific binding of 3H-5HT to T cell blasts was detectable and saturable. About 80% of 3H-5HT bound was displaced by 100 ⁇ M 5HT. Binding saturated at 1-3 ⁇ M 5HT. At saturation, 200-250 fmol 5HT bound per 10 T cells. Binding of 5HT saturated between 1-3 ⁇ M 5HT. The Kd, determined by Scatchard analysis was 180 nM (average of three separate experiments).
  • the inset to Figure 6A shows the Scatchard analysis of the binding data. Binding data from three separate experiments yielded an average dissociation constant of 200 nM.
  • Figure 6B shows binding of ketanserin to T cell blasts. Binding was saturable and T cell blasts expressed similar numbers of ketanserin and 5HT receptors. Specific binding of ketanserin was determined in the presence or absence of 100 ⁇ M 5HT.
  • the inset to Figure 6B shows the Scatchard analysis of ketanserin binding data which yields an average dissociation constant of 400 nM.
  • lymphocytes In lymphocytes, elevation of intracellular cAMP is generally associated with the inhibition of lymphocyte proliferation and lymphocyte effector function (57). Conversely, elevation of intracellular Ca++ is generally found to be required for activation of lymphocytes by antigenic or mitogenic signals and subsequent lymphocyte proliferation and effector function (53). Results presented here show that the 5HTla receptor can mediate an increase in intracellular Ca++ in Jurkat cells. This should clearly enhance lymphocyte proliferation and effector function.
  • the 5HTla receptor also regulates adenylate cyclase and levels of cAMP in lymphocytes. Depending upon the activation state of adenylate cyclase 5HTla agonists may either elevate or suppress levels cAMP and may either enhance or suppress lymphocyte proliferation and function. Biological response of Jurkat cells and T cell blasts to 5HT, the specific 5HTla agonist, 80HDPAT, and the 5HTla antagonist, spiperone.
  • 80HDPAT with or without 100 nM spiperone. Results are expressed as the concentration of intracellular Ca++. cAMP was extracted from cells after various treatments described above and quantitative as described. All cells were pretreated with 10 ⁇ M forskolin to activate adenylate cyclase. Results are expressed as pmol cAMP/ million cells. Comparison of the biological and pharmacological responses of 5HT and the 5HTla specific agonist, 80HDPAT, in T cells.
  • H-TdR Half-maximal increase in incorporation was obtained was 15 ⁇ M 5HT. This was observed when 5HT was added on days 3-5 of the culture period, the time at which 5HT receptors are maximally expressed (see table V) . Addition of 5HT earlier in the culture period also resulted in a three-fold enhancement of proliferation but more 5HT (100 ⁇ M) was required to achieve this effect.
  • 5HT early in the culture period reduces the effective concentration of 5HT in the culture medium by day 3.
  • 5HT, ketanserin (5HT2 receptor antagonist) and -methyl serotonin (5HT2 receptor agonist) stimulated T cell proliferation by about three-fold at concentrations between 10-30 ⁇ M.
  • 5HT2 receptor antagonists Two other 5HT2 receptor antagonists, ritanserin (5HT2 selective) and mianserin (5HT2 and 5HTlc selective) failed to stimulate T cell proliferation. In fact ritanserin was somewhat inhibitory at the higher concentrations.
  • CD4+ and CD8+ T cells proliferate in cultures of PBL stimulated with PWM.
  • CD8+ lymphocyte makes up the major percentage of proliferating cells on day 7.
  • Results in figure 5 also show that the ability to stimulate proliferation of PBL cultures with 5HT or 5HT receptor ligands is lost if CD8+ lymphocytes are eliminated by antibody and complement treatment.
  • Results in table 9 show that inhibition of 5HT synthesis by p-chlorophenylalanine (pCPA) also partially inhibited proliferation of cells stimulated by PWM. Addition of 5HT or ketanserin to these cultures reversed the inhibitory effects of pCPA. Table 9
  • CD8+ suppressor T cells make up the largest percentage of proliferating cells in cultures of PBL stimulated with PWM. Since inhibition of 5HT synthesis by pCPA inhibited proliferation in PWM stimulated cultures, it suggested that pCPA may also inhibit expression of suppressor cell function. These results are shown in figure 11. Cultures of PBL were stimulated with PWM in the presence or absence of pCPA to reduce 5HT content. Some cultures were also treated with ketanserin to provide an agonist for the 5HT receptor to possibly reverse the effects of pCPA. After 7 d, cultures were washed, treated with mitomycin C and added to fresh cultures of CD4+ T cells stimulated with PWM to test for suppression cell activity.
  • T cell mitogens such as PHA or OKT3 stimulate proliferation of both CD4+ and CD8+ T cells but do not result in the expression of suppressor cell activity by CD8+ T cells within the first week of culture.
  • PBL stimulated with either 0KT3 or PHA lose their 5HT content within 72 hrs while cultures stimulateded with PWM retain 5HT content for at least 7 d.
  • Addition of pCPA to cultures stimulated with either PHA or OKT3 did not inhibit proliferation (not shown).
  • PBL were stimulated with OKT3 in the presence or absence of the T cell 5HT receptor agonist, ketanserin (5HT receptor antagonist), and tested for suppressor cell activity after 5 days of culture.
  • T cell blasts harvested from cultures stimulated with OKT3 lacked detectable suppressor cell activity.
  • 5HT as a co-mitoqen for CD8+ T lymphocytes. Proliferation of CD8+ T lymphocytes in response to PWM requires CD4+ T cells. The three-fold enhancement of proliferation of PBL cultures caused by 5HT or 5HT receptor ligands was lost after eliminating CD8+ T cells. T erefore, it seemed possible that 5HT may serve as a co-mitogen for CD8+ T cells and stimulate proliferation in the presence of PWM. These results are shown in Figure 9. Cultures of CD8+ T cells failed to proliferate in the presence of PWM or in the presence of PWM and IL2. By contrast, cultures containing CD8+ T cells and PWM proliferated in the presence of either 5HT or ketanserin.
  • the level of proliferation was comparable to that when CD4+ T cells were added to cultures of CD8+ T cells. Addition of 5HT or ketanserin alone did not stimulate proliferation of CD8+ T cells. Taken together, these data suggest that 5HT is a co-mitogen for CD8+ suppressor T cells. Modulation of cAMP levels in activated T cells by 5HT. Two signal transduction pathways which have been linked to specific 5HT receptor subtypes are modulation of adenylate cyclase and activation of phospholipase C. Activation of phospholipase C results in elevation of inositol phosphates and increase in intracellular Ca++ concentration.
  • T cell blasts did not respond to 5HT by altering inositol phosphate levels and intracellular Ca++ concentrations
  • intracellular cAMP levels were compared in the presence and absence of 5HT.
  • Three cell types were compared: resting T cells, T cell blasts and Jurkat cells, and cAMP levels were compared in the presence and absence of forskolin. These results are shown in Table 8. Resting T cells did not change intracellular cAMP levels in response to 5HT where as activated T cells increased cAMP by over 2 fold in response to 5HT (5 ⁇ M) .
  • Jurkat cells did not alter cAMP levels in response to 5HT.
  • Forskolin activates adenylate cyclase and causes an increase in intracellular cAMP.
  • Forskolin increased cAMP concentration in resting T cells, activated T cells and Jurkat cells.
  • Forskolin-dependent increase in cAMP levels was inhibited by addition of 5HT to activated T cells but not to resting T cells or to Jurkat cells.
  • Figure 2 shows the concentration of 5HT required to cause an increase in cAMP levels in activated T cells, or in the presence of forskolin, a decrease in cAMP levels in activated T cells. Changes in cAMP levels were proportional to 5HT concentrations between 30 nM and 3 ⁇ M.
  • the half-maximal response was between 200-800 nM 5HT which was similar to the Kd obtained from the binding data.
  • Addition of PMA also results in an increase in cAMP levels in T cell blasts or in Jurkat cells.
  • 5HT failed to inhibit the PMA-dependent increase in cAMP in either cell type.
  • Table 9 Stimulation of proliferation of CD8+ T cells by 5HT. Changes in intracellular cAMP concentrations are known to affect the function and proliferations of activated T cells (53). Based on the above results, 5HT may regulate intracellular cAMP levels differently in different subpopulations on activated T cells, or in activated T cells exposed to different stimuli.
  • Results in Table VII compare changes in cAMP concentrations in T cell blasts initially stimulated with PHA, OKT3 or PWM.
  • T cells were harvested during peak proliferative responses and assayed for cAMP content in the presence or absence of 5HT. Cultures were also incubated with 5HT for the final 48 hr of culture to determine any affect on proliferation. 5HT caused a 2-fold increase in cAMP levels in T cells stimulated with PHA, a slight increase in cAMP in cells cultured with OKT3, but caused a 60% decrease in cAMP levels in T cells stimulated with PWM. Proliferation responses of the T cell cultures with the different stimuli in the presence or absence of 5HT appeared to reflect their levels of intracellular cAMP.
  • 5HT slightly inhibited proliferation of T cells in response to PHA but stimulated proliferation of T cells in response to PWM by over 3-fold. Additionally, elimination of CD8+ T cells before stimulation with PWM eliminated the co-mitogenic effect of 5HT in these cultures. Thus, 5HT can either enhance or suppress T cell proliferation, apparently by requlating cAMP levels. The direction of the response seems to depend upon the mitogenic stimulus and the T cell subpopulation responding to that stimulus. Results in Figure 3 show that the proliferative response of PWM activated T cells to 5HT is fairly rapid. Increases in proliferation in the presence of 5HT were detectable within hours and reached maximum levels with 40 hours. Levels of cAMP were decreased to 60% of control with 30 min and to 40% of control over the next 40 hr of culture.
  • Results in Figure 4 show the concentration dependence of the co-mitogenic effect of 5HT on PWM-activated T cells.
  • 5HT was added to cultures 48 hr before assay of proliferation. Maximal stimulation of proliferation was observed at 30 ⁇ M 5HT and the IC50 was 10 ⁇ M 5HT.
  • 5HT 5HT to T cells
  • Stimulation of T cell proliferation with OKT3 or PHA yields T cell blasts of both CD4 and CD8 phenotypes while stimulation of cultures of T cells with PWM yields T cell blasts primarily of the CD8 phenotype.
  • Responses of these two types of cultures to 5HT is also different.
  • Addition of 5HT to cultures stimulated with PHA or OKT3 results in elevation of cAMP and slight inhibition of T cell proliferation.
  • addition of 5HT to cultures stimulated with PWM causes a 60% decrease in cAMP levels and a 3-4 fold increase in T cell proliferation.
  • Cells proliferating in these cultures are primarily CD8+ T cells as measured by FMS (not shown) and exhibit suppressor cell but not cytotoxic activity. Elimination of CD8+ T cells eliminates the co-mitogenic effect of 5HT in these cultures.
  • 5HT is also a growth factor for certain other typed of cells such as smooth muscle cells or certain types of fibroblasts (46,47). This suggests that the growth factor properties of 5HT are not limited to cells of the immune system.
  • T cells express receptors for 5HT upon activation and that 5HT can influence the proliferation and function of activated T cells. It is well established that 5HT is a major component of the secretory granules of platelets and should be released at sites of inflammation (2-4). Thus 5HT should be present at sites of inflammation to influence T cell function. More recently it has been shown that purified resting T cells also contain 5HT (50). T cells release 5HT into the media in response to IFN gamma. Under these conditions, the concentration achieved in media is approximately 300 nM/ml/10 T cells. This is very similar to the Kd of the T cell 5HT receptor. If 5HT is released by T cells, it could play an important role in regulation of T cell function by T cells.
  • Rheumatoid arthritis is a major disease of a large group of rheumatic diseases.
  • Rheumatic diseases with an autoimmune component include rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome and Behcet's disease.
  • the arthritis of rheumatoid arthritis can result in destruction of the joint with consequent deformity.
  • the disease is not confined to joints; vasculitis, caused by immune complexes, can involve the skin, the eye, and the lung.
  • vasculitis caused by immune complexes, can involve the skin, the eye, and the lung.
  • the arthritis results from a complex interaction of synovial cells with various cellular elements (and their soluble products) that infiltrate from the circulation into the synovial lining of joints. This leads to massive proliferation and activation of synovial cells.
  • the properties of synovial cells in tissue culture have been likened to those of transformed cells.
  • a form of experimental arthritis is adjuvant arthritis, which is a purely T cell-mediated autoimmune disease.
  • This form of arthritis can be induced in susceptible strains of rats (e.g. Lewis rats) by injection of Mycobacterium tuberculosis in oil (complete Freund's adjuvant).
  • Mycobacterium tuberculosis in oil (complete Freund's adjuvant).
  • a nonpeptide of a Mycobacterium tuberculosis antigen contains the epitope recognized by T cells mediating adjuvent arthritis.
  • T cells from patients with rheumatoid arthritis respond to this shared epitope. Paul, supra. Protocol: Adjuvant Arthritis
  • CFA Complete Freund's Adjuvant
  • Drugs are dissolved or suspended in water, physiologic saline or a mixture of either of the latter in 2% polyethylene glycol 400/0.1% Tween 80 and administered by normal routes (orally, intravenously or intramuscularly) either prior to or after establishment of disease.
  • Adjuvant arthritis is clearly mediated by T cells and activated T cells clones have been isolated which can transfer the disease to a naive animal.
  • Ritanserin inhibits development of the disease at 10 or 30 mg/kg. Ketanserin does not prevent development of the disease.
  • ⁇ Difference in activity may be explained by slightly different dosing schedule.
  • Materials Purified recombinant human IFN was purchased from Amgen (Thousand Oaks, CA) and diluted in media before use. 5-OH-tryptophan, 5HT, tryptophan, 5-OH-indoleacetic acid, and melatonin were obtained from Sigma and p-chlorophenylalanine (pCPA) was obtained from Research Biochemicals, Inc. (Natick, MA). RPMI 1640 media, fetal calf serum (FCS), sodium pyruvate and sodium glutamate were from GIBCO. Flat-bottom multi-well tissue culture dishes were from Becton Dickinson or from Costar.
  • Solvents for HPLC were from Aldrich and were HPLC grade.
  • Cell Cultures The human cervical carcinoma cell line, ME-180, was obtained from American Type Culture Collection (Rockville, MD) and was maintained in tissue culture flasks in RPMI 1640 medium with 10% FCS without antibiotics in a humidified atmosphere of 5% CO, in air at 37°C.
  • ME-180 cells were plated in
  • the gradient 5 was linear from 100% A to 100% B over 25 min.
  • the retention times of known standards were as follows: 5HT, 3.50 min; 5-OH-tryptophan, 4.77 min; tryptophan, 9.11 min; 5-OH-indoleacetic acid, 12.51 min; and melatonin, 21.69 min.
  • Identification of peaks in cell extracts with 0 known standards was achieved by mixing known amounts of standards with cell extracts and showing identity of peaks on the chromatograms.
  • Recovery of tryptophan and 5HT from cell extracts was greater than 90% as determined by mixing known amounts of standards with cells before initiating the extraction procedure. Experiments quantitating levels of 5HT and tryptophan were performed three times each with similar results.
  • 5Htp is greater than 500-fold more effective than tryptophan at inhibiting IFN activity against ME-180 cells ( Figure IB) .
  • 5Htp is a precursor for 5HT synthesis and is not used for protein synthesis. This raises the possibility that inhibition of 5HT synthesis may be an important result of tryptophan depletion in cultures of cells exposed to IFN.
  • Results in Figure 2 compare IFN -mediated inhibition of cell proliferation. Optimal results were obtained when compounds were added on day one and two of the three day culture. Under these conditions only 5Htp blocked IFN activity.
  • Other 5Htp metabolites, 5HT, melatonin, N-AC-5HT or 5-OH-indoleacetic acid also products of 5Htp metabolism, failed to block IFN-mediated inhibition of cell proliferation.
  • IFN depresses intracellular levels of both tryptophan and tHT.
  • the above results raise the possibility that loss of tryptophan in culture media results in loss of 5Htp or its metabolites, such at 5HT, and this leads to inhibition of cell proliferation.
  • Figure 3 shows the level of intracellular tryptophan and 5HT in cells after varying periods of time in culture with IFN. Cells contained both tryptophan and 5HT; both were lost upon culture with IFN. 5Htp was undetectable in cell extracts. Synthesis of 5Htp is the rate limiting step in 5HT synthesis and 5Htp does not generally accumulate in cells.
  • 5Htp restores levels of 5HT and cell proliferation in IFN treated cells.
  • Tumor cells were cultured with inhibitory amounts of IFN in the presence or absence of 5Htp to determine if 5Htp restored 5HT levels (Table 5).
  • Treatment with IFN resulted in a decrease in 5HT to undetectable levels.
  • levels of 5HT were maintained.
  • cultures of cells with IFN and 5Htp contained four-fold higher levels of 5HT than untreated cultures. Addition of 5Htp also restored cell proliferation in these cultures. 5Htp does not prevent the loss of tryptophan from media in the presence of IFN.
  • ME-180 cells The above results suggest that lowering intracellular 5HT should inhibit proliferation of ME-180 cells. Therefore, ME-1870 cells were cultured with pCPA, a specific inhibitor of tryptophan hydroxylase, to determine if this resulted in loss of intracellular 5HT and inhibition of cell proliferation. The results are shown in Table 6. Culture of ME-180 cells for 48 hr with pCPA resulted in loss of tHT and inhibition of cell proliferation but did not cause a loss in extracellular tryptophan. Loss of 5HT and inhibition of cell proliferation was prevented by addition of 5Htp.
  • indoleamine 2,3 dioxygenase catalzyed oxidation of tryptophan or inhibition of tryptophan hydroxylase resulted in inhibition of tumor cell proliferation.
  • tumor cell proliferation was recovered by restoring 5HT levels with 5Htp.
  • Serotonin was found to be present in neoplastic or tumor cells and the 5HT2-like receptor was found to be present on tumor cells. It has been demonstrated that like activated T cells, tumor cell proliferation can be regulated by serotonin receptor agonists and antagonists as well as by inhibition of serotonin synthesis.
  • serotonin (5-hydroxytryptamine, 5HT) is synthesized by hydroxylation and decarboxylation of tryptophan and is stored in granules (35-36). It is released in response to appropriate stimuli and binds to specific receptors on neighboring cells activating second messenger pathways (37-39).
  • the rate limiting step in serotonin synthesis is the level of tryptophan hydroxylase activity.
  • a specific enzyme inhibitor of tryptophan hydroxylase, p-chlorophenylalanine (pCPA) has been employed to deplete serotonin levels within cells (40-41).
  • Human cervical carcinoma cells (ME-180) were treated with pCPA for 48 hr in the presence or absence of 5-OH-tryptophan and analyzed for 5HT content or for rates of cell proliferation.
  • Cultured cells contained 13 ⁇ 2 pmol/mg protein 5HT which was reduced to 4 ⁇ 1 pmol/mg protein 5HT after treatment with pCPA.
  • media from cultured cells contained 44 nM 5HT which was reduced to 10 nM by culturing with pCPA.
  • Addition of 5-OH-tryptophan restored 5HT levels to that of control cells.
  • Treatment of ME-180 cells with pCPA also reduced H-TdR incorporation by 70%. Inhibition was also largely reversed by 5-OH-tryptophan (Table VIII).
  • ritanserin a 5HT2 receptor antagonist 40
  • ritanserin inhibited tumor cell proliferation by 70-95% (five of six lines tested) at concentration between 10-50 ⁇ M but did not inhibit proliferation by normal diploid cells.
  • Lower concentrations of ritanserin did not inhibit proliferation by tumor cells.
  • Addition of 5HT partially reversed the inhibitory effects of ritanserin (Table X) .
  • Proliferation by the one tumor cell line, Molt-4 which was not inhibited by pCPA, was also not inhibited by ritanserin.
  • pCPA lowered concentrations of 5HT in cell extracts
  • ritanserin actually increased 5HT content in cell extracts.
  • extracts from ME-180 cells contained 15.5 pmol/mg protein of 5HT while extracts from cultures treated with ritanserin contained 58.8 pmol/mg protein of 5HT.
  • both inhibitors of 5HT synthesis as well as 5HT receptor antagonists inhibited proliferation by tumor cells but not by normal diploid cells.
  • a number of ligands which bind 5HT receptors were tested for their ability to prevent ritanserin-mediated inhibition of tumor cell proliferation. These results are shown in figure 14.
  • Ketanserin and pelanserin also 5HT2 receptor antagonists, mianserin, a 5HTlc receptor antagonist and 8-OH-DPAT and propranolol, a 5HT1 receptor agonist and antagonist, respectively, partially prevented inhibition of proliferation by ritanserin in a concentration dependant manner.
  • 5HT2 receptor antagonists mianserin, a 5HTlc receptor antagonist and 8-OH-DPAT and propranolol, a 5HT1 receptor agonist and antagonist, respectively.
  • ritanserin, ketanserin and pelanserin are all 5HT2 receptor antagonists with similar pharmacological and biochemical properties but ritanserin inhibits tumor cell proliferation and ketanserin and pelanserin prevent inhibition by ritanserin.
  • 5HT has been shown to be a growth factor for both quiescent fibroblasts as well as smooth muscle cells (45-47). In these instances exogenous as opposed to endogenous 5HT was employed to stimulate DNA synthesis. These results are consistent with many current notions of tumorigenesis where normal diploid cells may become transformed by acquiring the ability to produce their own growth factors or growth factor receptors (48-49). Production of 5HT and 5HT receptors may be one way which normal cells acquire a permanent transformed phenotype. Whether tumor cells synthesize serotonin and require serotonin for proliferation in vivo is an important question which is unanswered.
  • Inhibition of 5HT synthesis inhibits proliferation by tumor cells but not by normal diploid cells.
  • Different human cell lines were obtained from ATCC. Their culture conditions have been described (50). Sera were dialyzed before use to remove 5HT.
  • ME180 cervical carcinoma
  • BT20 breast carcinom
  • HT29 colon adenocarcinoma
  • U937 histocytic lymphoma, monocyte lineage
  • Jurkat T cell lymphoma
  • MOLT-4 acute lymphoblastic leukemia
  • endothelial cells primary cultures
  • synovial fibroblasts primary cultures
  • lung fibroblasts MRC-5; long term line
  • Other conditions were as in Table 11.
  • Table 13 Inhibition of proliferation of tumor cells by a 5HT receptor antagonist and reversal by 5HT.
  • Tumor cell lines and normal diploid cells were cultured for 48 hrs in the presence or absence of 30 ⁇ M retanserin (a 5HT2 receptor antagonist which is active against a 5HT2 site at concentrations of 1-10 nM) with or without 1 ⁇ M 5HT. Addition of 5HT did not affect control proliferation. Cultures were set up in triplicate and the standard error was less than 10% (data not shown) .
  • antibodies "types" have in common is the ability to recognize an epitope or epitopes specific to the novel 5HT2-like receptor.
  • purified receptor With the identification of the novel 5HT2-like receptor, purified receptor can be used as a probe for
  • SUBSTITUTE SHEET delayed-type hypersensitivity Serotonin-2 receptor antagonists inhibit contact sensitivity by an effect of T cells. J. Immunol. 1989: 3171.

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Abstract

Procédé de régulation de la prolifération de cellules néoplasiques présentant un récepteur 5HT1a, consistant à introduire une quantité suffisante d'agonistes ou d'antagonistes pour accroître ou réduire la prolifération cellulaire. La prolifération cellulaire peut être régulée (1) par l'intermédiaire du récepteur 5HT et/ou (2) par l'intermédiaire de l'inhibition de la synthèse de la sérotonine.
EP91915814A 1990-09-04 1991-09-04 Regulation de la proliferation de cellules neoplasiques par l'intermediaire d'un nouveau recepteur 5ht1a Withdrawn EP0555231A1 (fr)

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WO1996001107A1 (fr) * 1994-07-06 1996-01-18 Bo Arne Hofmann Emploi d'agents pharmaceutiques retablissant, ameliorant ou traitant la deficience immunitaire et notamment ameliorant ou traitant certains troubles immunitaires lies a des infections par le vih
AU2880495A (en) * 1994-07-06 1996-01-25 Bo Arne Hofmann Use of pharmaceutical agents for alleviation or treatment of the immune dysfunction related to infection with human immunodeficiency viruses (hiv) or related viruses
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WO1992004014A2 (fr) 1992-03-19
AU8848291A (en) 1992-03-30
JPH06500775A (ja) 1994-01-27
CA2090689A1 (fr) 1992-03-05
EP0547172A1 (fr) 1993-06-23
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WO1992004014A3 (fr) 1992-05-14

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