EP0547172A1 - REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a - Google Patents

REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a

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EP0547172A1
EP0547172A1 EP91918533A EP91918533A EP0547172A1 EP 0547172 A1 EP0547172 A1 EP 0547172A1 EP 91918533 A EP91918533 A EP 91918533A EP 91918533 A EP91918533 A EP 91918533A EP 0547172 A1 EP0547172 A1 EP 0547172A1
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cells
receptor
cell
proliferation
5ht1a
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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

  • TITLE Regulation of T-Cell Proliferation via a Novel 5HT1a Receptor
  • 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 5HT1a family of receptors. For Jurkat cells and activated T cells this is based on the following criteria: binding studies with the 5HT1a receptor specific agonist (8OHDPAT), studies on signal transduction, northern analysis with specific human 5HT1a oligionucleotide probes. Resting T cells did not express 5HT1a 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 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 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).
  • T lymphocytes The activation of resting T lymphocytes is critical to most immune responses since cellular activation allows these cells to exert their regulatory or effector activities.
  • relatively quiescent cells undergo complex changes involving cell differentiation and proliferation.
  • 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 participate in the complex cell-cell interaction which occurs during antigen presentation, 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 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 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.
  • 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 mimotopes, small peptide ligands, that bind to either the 5HT2-like receptor or to antibodies to the 5HT2-like receptor.
  • mimotopes small peptide ligands
  • Such 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 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 5HT1c or 5HT2 receptors (by gene transfeetion) in normal diploid murine fibroblasts results in the acquisition of a transformed phenotype in these cells.
  • transfected cells After introducing cDNA's encoding these receptors, 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, to grow in soft agar and to form tumors in nude mice. This raises the possibility that synthesis of serotonin and aberrant expression of 5HT receptors may play an important role in tumorigenesis.
  • Normal cells may acquire a transformed phenotype by acquiring the ability to synthesize serotonin and/or serotonin receptors which can transduce second messenger pathways. Results presented here show (1) proliferating cells contain serotonin, (2) inhibition of serotonin synthesis inhibits cell proliferation by tumor cells but not by normal cells, and (3) proliferation of tumor cells but not normal cells is inhibited by certain serotonin receptor antagonists.
  • 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.
  • This 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 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.
  • 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 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 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 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 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 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.
  • FIG. 1 is a graph demonstrating 5HT binding to Jurkat cells.
  • Jurkat cells (1 x 10 6 ) were incubated with the indicated concentrations of ( 3 H)5HT for 6 min at 4oC in the absence (total binding) or presence of 50 ⁇ M 5HT (nonspecific binding). Specific binding ( ) is expressed as the difference between these two values.
  • Figure 1A shows the results graphed on a Scatchard plot.
  • Figure 2 is a graph demonstrating kinetics of association and dissociation of ( 3 H)5HT to Jurkat cells.
  • A Time course of association of ( 3 H)5HT to Jurkat cells. Conditions were as described in Materials and Methods except that time of incubation was varied ( ).
  • B Time course of dissociation of bound ( 3 H)5HT ( ) from Jurkat cells.
  • 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. OKT3 (1 ⁇ g/ml) or 5HT
  • the relative intracellular calcium concentration was calculated from the ratio of violet fluorescence (calcium bound indo-1) to blue fluorescence (calcium-free indo-1) and plotted as a function of time.
  • Figure 4 is a graph demonstrating concentration dependence of 5HT mediated increases in intracellular Ca 2+ .
  • Jurkat cells, loaded with indo-1 were incubated with 5HT at 37°C for 2 min.
  • Percent of cells with increased intracellular CA 2+ ( ⁇ ) and total intracellular CA 2+ concentration ( ⁇ ) was determined by measuring the increase in fluorescence with the use of FACSTAR Plus as described in Materials and Methods.
  • Percent positive cells contained 375 to 4 25 nM Ca 2+ and negative cells contained 150 to 175 nM Ca 2+ .
  • Figure 5 is a graph demonstrating 5HT-mediated increase in phosphatidylinositol turnover.
  • Jurkat cells were labeled with ( 3 H)inositol for 48 h. Labeled cells were incubated with 3 ⁇ M 5HT ( ⁇ ) or 1 ⁇ g/ml OKT3 ( ⁇ ) for the indicated periods of time before harvest and analysis of IP levels by anion exchange chromatography.
  • 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.
  • FIGS 6A-1 and 6B-1 depict Scatchard analysis of the binding data from Figures 6A and 6B, respectively.
  • 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.
  • Figures 13A and 13B are graphs of properties of the serotonin receptor on tumor cells.
  • FIG. 13A shows a Scatchard analysis of binding data with 5HT ( ) and ketanserin ( ⁇ ). The Kd was 130 nM.
  • B Cells were incubated at 0°C for 6 min in the presence of
  • FIG. 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 ( ), pelanserin ( ⁇ ), Ketanserin ( ), mianserin ( ⁇ ), 80H-DPAT( ⁇ ), propranolol ( ⁇ ).
  • 5HT 5HT
  • pelanserin ⁇
  • Ketanserin мер ⁇
  • mianserin mianserin
  • 80H-DPAT( ⁇ ) 80H-DPAT( ⁇ ), propranolol
  • Results are expressed as the percent of control incorporation of 3 H-TdR into ME180 cell cultures in the absence of ritanserin or other ligands. Incorporation of 3H-TdR by ME180 cells was
  • 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 (0) 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 ( ⁇ ) or
  • FIG 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 (0), 5HT (I), melatonin ( ⁇ ), N-methyl serotonin ( ⁇ ) 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.
  • 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 (0) or absence (0) of 100 u/ml IFN , harvested and analyzed for 5HT content and tryptophan content by HPLC; UPPER GRAPH: 5HT; LOWER 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 ( , ⁇ ) and in the presence ( ) or absence ( ⁇ ) 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. Results are expressed as incorporation of 3 H-TdR after a 6 hr pulse on day 2.
  • 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 and were maintained in continuous culture in RPMI 1640 medium supplemented with 10% FCS without antibiotics but with 1 mM glutamine.
  • Binding Assay Jurkat cells were harvested from log phase cultures and were incubated at 3.0x10 6 /ml in a total volume of 600 ⁇ l or RPMI 1640 at 0-4°C in the presence of 3H-5HT with or without various 5HT agonists or antagonists (see text for details) for 6 min. Binding equilibrium was reached during this incubation period. Displacement experiments were performed by incubating cells with 3 HH-5HT, followed by incubation with unlabeled agonists or antagonists as described in the text. Cells were collected onto glass fiber filters and tubes and filters were washed 3 times each with 4 ml of cold PBS.
  • Ascorbate 100 ⁇ M was included in the wash solutions when 125 I-LSD or 3 H-80H-DPAT binding was evaluated to reduce nonspecific binding to the filters (14-15). Washing of each filter took less than 20 sec. Total binding of 3 H-5HT to Jurkat cells was typically
  • Intracellular Ca++ measurements Mobilization of intracellular Ca++ was evaluated by the indo-1 method using a modified Becton Dickinson FACSTAR Plus (16-17). Briefly, Jurkat cells were incubated with 2 ⁇ M indo-1 in DMEM with 10 mM HEPES for 45 min. at 37°C, washed three 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. Approximately 5 sec. after data collection began, 5HT or OKT3 (see text for concentrations) was added directly to the sample and data acquisition continued for a total of 4 minutes.
  • 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 in list mode was subsequently processed to yield a ratio of violet fluorescence (395/20 nm) to blue fluorescence (485/22 nm) as a function of time. Calcium concentration was calculated using the formula:
  • Kd is the dissociation constant (250 nm); R, Rmin, and Rmax are the fluorescence intensity ratios at resting, zero, and saturation calcium concentrations, respectively; and Sf2/Sb2 is the ratio of fluorescence intensity of the calcium-free and calcium-bound dye (18).
  • Cells were suspended in media without FCS at 1x10 6 /ml at 37°C, treated with 5-HT as described in the text, harvested at various times by centrifugation. Extracts were neutralized with 7 N KOH and analyzed for levels of IP by anion exchange chromatography as previously described (19). Duplicate samples were within 5% and experiments measuring inositol phosphate levels were performed three times with similar results.
  • PBMC peripheral blood mononuclear cells
  • PWM and PHA were obtained from GIBCO (OKT3 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.
  • IL-2 activity is defined as the amount required to cause 50% maximal proliferation of the IL-2 dependent cell line
  • PBMC, CD4+PBMC or CD4+ cells were cultured at 5x10 5 cells/ml in RPMI-1640 media supplemented with 10% FCS without antibiotics for 3 to 8 days in flat bottom 96-well microculture plates (Becton
  • Binding assays were performed as described on p15-16 for Jurkat cells.
  • 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 for cell proliferation by addition of 3 H-TdR. Media and cells were separated, 5HT was extracted and protein was precipitated with 70% ethanol, and samples were concentrated with a Speed-vac. Samples were analyzed for
  • 5HT content by HPLC (Hewlet-Packard) equipped with a fluorescence detector (Beckman) with a 280 nm excitation filter and a 360nm emission filter.
  • the two mobile phases consisted of A: 50mM triethylamine adjusted to pH
  • 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). Results in figure 1 show that binding of 3 H-5HT to Jurkat cells was detectable and that binding was saturable.
  • Binding of 5HT reached a maximum at 1-2 ⁇ M 5HT.
  • Figure 1A shows the Scatchard plot of the binding data.
  • 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 prevented binding of 3 H-5HT to both membrane preparations and to intact cells. The major difference between binding of 5HT to membrane preparations or to intact cells was in the calculated number of receptors/cell . Intact cells expressed 80,000 receptors/cell whereas calculations derived from binding data from isolated membrane preparations yielded 30,000 receptors per cell. At this point it is not certain where this difference arises from. However, since binding data are compared to the biological response of intact cells to 5HT as a function of 5HT concentration, only results of binding of 5HT to intact cells will be reported here.
  • the ratio of ks divided by k1 provides a calculated Kd of 180 nM. This value agrees reasonably well with the Kd calculated by Scatchard analysis.
  • Cell surface receptors for 5-HT have been classified into several subtypes on the basis of pharmacological as well as functional properties (12-13). Binding of 5-HT to 5-HT1a or 5-HT1b sites results in inhibition of stimulated adenylate cyclase activity while binding to 5-HT1c 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
  • 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).
  • 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.
  • Jurkat T-cells were labeled for 48 hrs. with 3 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 agonist, and 5HT inhibited binding of 3 H-5HT to Jurkat cells. Concentrations which inhibited 50% of specific binding of 3 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).
  • 1 25 I-LSD, 3 H-ketanserin and 3 H-DOB have been used to label 5HTlc or 5HT2 sites in the central nervous system.
  • 3H-80HDPAT has been used to label 5HT1a 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 100 ⁇ M 5HT.
  • 3H-ketanserin and 3 H-5HT labeled Jurkat cells whereas 1 25 I-LSD and 3 H-DOB failed to label Jurkat cells under the conditions employed.
  • 3 H-80HDPAT also failed to label
  • 5HT receptor agonists and antagonists have affinity constants in the range of 1-5 nM for their specific receptor classes.
  • b EC50's were determined by incubating Jurkat cells with various concentrations of 5HT2 agonists and following change in Ca++ concentration as a function of time. The rate of change of Ca++ is linear for the first 60 sec. and is complete 2-3 min. after addition of 5HT agonists.
  • Jurkat cells (1x10 6 /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 following designations have been employed; the 5HT1 family consists of 5HT1a, 5HT1b, and 5HT1d; the 5HT2 family consists of 5HT1c, 5HT2a and 5HT2b, and the 5HT3 family presently consists of only one member. cDNA's encoding 5HT1a, 5HT1c and 5HT2 receptors have been identified and all encode proteins which are G protein-linked receptors with seven membrane spanning units (23-25).
  • binding of 5HT to 5HT1a 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 5HT1a sites. Two of these are 80H-DPAT and ipsapirone. Binding of 5HT to 5HTlc or 5HT2 sites results in increased phosphatidylinositol turnover and intracellular Ca++ mobilization.
  • a number of 5HT2 antagonists have been identified which interact with 5HT1c and 5HT2 sites with equivalent affinities and examples include mesulergine and mianserin. Other 5HT2 antagonists include ketanserin, ritanserin, and pelanserin.
  • 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. In these tissues, phosphatidylinositol turnover is only inhibited by very high concentrations of ketanserin (compare 1-10 ⁇ M to the normal affinity of ketanserin for 5HT2 sites of 1-10 nM (27-28).
  • 5HT3 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 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 sites per cell.
  • 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 Kd for 5HT binding to Jurkat cells.
  • Binding of 3 H-5HT and 5HT mediated Ca++ mobilization were not affected by the specific 5HT1a agonist 80H-DPAT or by the specific 5HT3 ligand ICS-205930. Further, 5HT or 80H-DPAT did not modulate cAMP concentrations in Jurkat cells (data not shown). These data are consistent with binding of 5HT to the 5HT2 receptor family. Additionally, alpha methyl serotonin maleate, a 5HT2 agonist, displaced bound 5HT and stimulated Ca++ mobilization in Jurkat cells. However, ketanserin bound to Jurkat cells with only weak affinity (Kd of 100 nM) and no specific binding of 125 I-LSD (displaced by 5HT) to Jurkat cells could be detected.
  • mianserin, mesulergine, pelanserin, and ritanserin did not inhibit 5HT binding to Jurkat cells at concentrations ⁇ 20 ⁇ M and did not inhibit 5HT mediated Ca++ mobilization.
  • Ketanserin only displaced 5HT and inhibited Ca++ mobilization at high concentrations (50% at 10 ⁇ M.
  • 3 H-DOB, a specific 5HT2a ligand did not label Jurkat cells. Effects of 5HT antagonists were equivalent when 5HT binding was compared to Ca++ mobilization studies. Taken together these data suggest that the 5HT receptor on Jurkat cells is not a 5HT1c, 5HT2 or 5HT2b receptor.
  • 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 in 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 3 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).
  • T cell blasts bound 224 fmol 5HT per 106 cells which represents a site density of 120,000 receptors per cell. These results are shown in Figure 6A.
  • Figure 6A-1 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.
  • Figure 6B-1 shows the Scatchard analysis of ketanserin binding data which yields an average dissociation constant of 400 nM. Taken together, these data support the notion that their is a single receptor on T cell blasts which binds both 5HT and ketanserin within these concentration ranges. Competition experiments with various 5HT receptor ligands showed that 5HT, ⁇ -methyl serotonin and ketanserin could compete with H-5HT for binding to T cells, Figure 7. IC50's were 0.1 ⁇ M, 0.06 ⁇ M and 20 ⁇ M, respectively.
  • 5HT receptor ligands such as 80H-DPAT (specific for 5HT1a receptors), ICS-205930 (specific for 5HT3 receptors), mianserin (specific for 5HT1c and 5HT2 receptors) and spiperone (specific for 5HT2 receptors) did not compete with 5HT for binding to T cell blasts.
  • the properties of the 5HT receptor on T cell blasts are comparable to the properties of the 5HT receptor found on Jurkat cells and appear distinct from the well characterized receptors found in nervous tissue.
  • T cell populations were evaluated for the presence of 5HT receptors.
  • Resting lymphocytes expressed low levels of receptors (24,000/cell) when compared to T cell blasts (150,000/cell).
  • Both CD4+ and CD8+ T cell lines expressed elevated levels of 5HT receptors (143,000 and 171,000 receptors/cell, respectively while CD3+,4-,8- T cell lines only expressed 53,000 receptors/cell which is only slightly more than that observed on resting lymphocytes.
  • expression of 5HT receptors was examined as a function of time after stimulation with different T cell mitogens. Increase in the number of receptors was linear for 72 hr after stimulation . Three T cell mitogens , PHA, PWM, or OKT3 , did not yield substantially different increases in the 5HT receptor number on T cells (Table 8).
  • 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 5HT1a receptor can mediate an increase in intracellular Ca++ in Jurkat cells. This should clearly enhance lymphocyte proliferation and effector function.
  • the 5HT1a receptor also regulates adenylate cyclase and levels of cAMP in lymphocytes. Depending upon the activation state of adenylate cyclase 5HT1a 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 5HT1a agonist, 80HDPAT, and the 5HT1a 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 5HT1a specific agonist, 80HDPAT, in T cells.
  • PBL contained 4 pmol 5HT/10 cells at culture initiation. This level was maintained for 4 d but was diminished by day 7.
  • stimulation of PBL with either OKT3 or PHA resulted in loss of over 75% of 5HT content within 72 hr, about the time 5HT receptors have achieved maximum levels.
  • Stimulation of PBL with PWM resulted in an initial increase in 5HT levels by 50% over the first 48 hr of culture and then a gradual decrease. These cells still maintained about 2 pmol 5HT/10 cells on day 7.
  • 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.
  • 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 OKT3 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. Therefore, it seemed possible that 5HT may serve as a co-mitogen for CD8+ T cells and stimulate proliferation in the presence of PWM.
  • 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.
  • 5HT stimulates elevation of inositol phosphates and intracellular Ca++ concentration. Therefore, changes in inositol phosphate levels and intracellular Ca++ were measured in T cell blasts (PHA) in response to 5HT and, as a positive control, in response to OKT3. Results in Table 7 summarize these experiments.
  • 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. In contrast to the case with forskolin, 5HT failed to inhibit the PMA-dependent increase in cAMP in either cell type. These results are shown in Table 9.
  • 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.
  • elimination of CD8+ T cells before stimulation with PWM eliminated the co-mitogenic effect of 5HT in these cultures.
  • 5HT can either enhance or suppress T cell proliferation, apparently by requlating cAMP levels.
  • 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 ummune complexes, can involve the skin, the eye, and the lung.
  • vasculitis caused by ummune 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).
  • mycobacterial peptidoglycans and proteoglycans in joint cartilage.
  • 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.
  • CFA Complete Freund's Adjuvant
  • extra heavy mineral oil 10 mg/ml heat killed Mycobacterium butyrium or Mycobacterium tuberculosis H37Ra.
  • female Lewis rats 200-225 g are given 0.1 ml injection of this adjuvant (100 ⁇ g/animal) subcutaneously into the right hind footpad (injected paw).
  • a primary inflammatory reaction occurs in the injected foot. This response subsides by day 5 and is followed on days 9-12 by a secondary, chronic inflammatory/arthritis response in both the injected foot and the contralateral non-injected left foot.
  • 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.
  • 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 2 in air at 37°C.
  • ME-180 cells were plated in complete media at 1x10 5 cells/ml, 100 ⁇ l/well in 96 well plates and were cultured in the presence or absence of varying amounts of IFN for 3 d.
  • cultures were pulsed with 1 ⁇ Ci 3 H-TdR for 5 hr and were harvested on filter paper. Cultures were performed in duplicate a minimum of three times with similar results; many were repeated as positive controls for subsequent experiments. Duplicates were within 10% of each other. Incorporation of 3 H-TdR was determined with a liquid scintillation spectrometer. Extraction and analysis of tryptophan, 5-OH-tryptophan, and 5HT.
  • 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 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 17B).
  • 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.
  • Two possible explanations for the ability of 5Htp to prevent IFN-mediated inhibition of cell proliferation are that it prevents the loss of tryptophan from media or that it restores intracellular
  • 5HT levels 5HT levels. Results in Figure 4 show that 5Htp does not inhibit loss of extracellular tryptophan in cultures of cells exposed to IFN. Control cultures of ME-180 cells did not consume significant amounts of tryptophan in media over the 72 hr culture period. By contrast, cultures of cells treated with IFN consumed 50% of tryptophan in 24 hr and 95% within 48 hr. The rate of loss of tryptophan in media for IFN treated cultures was not inhibited by addition of 5Htp. Under these conditions, levels of 5HT and cell proliferation were restored to control values.
  • Figure 5 compares proliferation of ME-180 cells in the presence of varying concentrations of tryptophan with or without 3 ⁇ M 5Htp. In the absence of
  • 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.
  • 5Htp restores 5HT levels and proliferation in IFN-treated ME-180 cells.
  • 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, 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. This is not to imply that these data are sufficient to define a new 5HT receptor subtype. Analysis of these receptors in much greater detail will be required to completely define these 5HT receptor types. Taken together these data raise the possibility that 5HT and 5HT receptors may be required for proliferation of certain tumor cell lines in tissue culture. Some of the characteristics of this proliferation is consistent with an autocrine pathway of growth factor action where a cell releases its own growth factors which bind to cell surface receptors and stimulate cell function (43-44).
  • 5HT is produced by tumor cells and that it is required for proliferation.
  • 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.
  • 5HT receptor antagonist 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).
  • purified receptor can be used as a probe for screening libraries of random peptide sequence to identify peptide that specifically bind to proteins.
  • antibodies generated against the purified receptor can be used as a probe.
  • Cwirla et al. Peptides on page: A vast library of peptides for identifying ligands, Proc.Natl. Acad. Sci. USA, Vol. 87, 6378-6382, August 1990.
  • methods of screening see Devlin et al.. Random Peptide Libraries: A Source of Specific Protein Binding Molecules, Science, Vol. 249, 404-406, 27 July 1990 and Scott and Smith, Searching for Peptide Ligands with an Epitope Library Science, Vol. 249, 386-390, 27 July 1990.

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Abstract

Le procédé décrit qui permet de réguler la prolifération de lymphocytes T activés présentant un récepteur de type 5HT1a, consiste à introduire une quantité suffisante d'agents agonistes ou antagonistes dans le but soit d'augmenter soit de diminuer la prolifération des lymphocytes T. Le moyen utilisé comme base de régulation de la prolifération des lymphocytes T peut être: (1) le récepteur 5HT, (2) l'inhibition de la synthèse de la sérotonine, et/ou (3) la stimulation par la sérotonine des sous-populations CD8+ de lymphocytes T activés.
EP91918533A 1990-09-04 1991-09-04 REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a Withdrawn EP0547172A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US57871090A 1990-09-04 1990-09-04
US578710 1990-09-04

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EP0547172A1 true EP0547172A1 (fr) 1993-06-23

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EP91918533A Withdrawn EP0547172A1 (fr) 1990-09-04 1991-09-04 REGULATION DE LA PROLIFERATION DE LYMPHOCYTES T VIA UN NOUVEAU RECEPTEUR, LE 5HT1a
EP91915814A Withdrawn EP0555231A1 (fr) 1990-09-04 1991-09-04 Regulation de la proliferation de cellules neoplasiques par l'intermediaire d'un nouveau recepteur 5ht1a

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EP91915814A Withdrawn EP0555231A1 (fr) 1990-09-04 1991-09-04 Regulation de la proliferation de cellules neoplasiques par l'intermediaire d'un nouveau recepteur 5ht1a

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EP (2) EP0547172A1 (fr)
JP (2) JPH06503816A (fr)
AU (2) AU8848291A (fr)
CA (2) CA2090689A1 (fr)
WO (2) WO1992004014A2 (fr)

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US5461054A (en) * 1993-12-09 1995-10-24 Bayer Aktiengesellschaft Anthracene-spiro-pyrrolindines
US5411960A (en) * 1993-12-09 1995-05-02 Bayer Aktiengesellschaft Substituted pyrroloanthracenes and -diones
US5409932A (en) * 1993-12-09 1995-04-25 Bayer Ag Piperazine-substituted pyrroloanthracenes
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
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
EP0813878B1 (fr) * 1996-06-17 2002-02-06 Mitsubishi Chemical Corporation Agent pour accélérer la lacrimation contenant un ligand de la sérotonine, particuliérement un composé aminoalkoxybibenzyle
WO2002078643A2 (fr) 2001-03-30 2002-10-10 Philadelphia Health And Education Corporation Immunomodulation et action sur des processus cellulaires relatifs aux recepteurs de la famille de la serotonine
KR101519028B1 (ko) * 2007-04-13 2015-05-11 써던 리서취 인스티튜트 항혈관형성제 및 사용방법
CN113599370B (zh) * 2021-08-03 2023-12-08 复旦大学附属肿瘤医院 8-oh-dpat及其衍生物在制备抗肿瘤药物中的应用

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WO1991002527A1 (fr) * 1989-08-21 1991-03-07 Beth Israel Hospital Association Procede et composition de traitement d'hypersensibilite cutanee, oculaire et des muqueuses, d'etats inflammatoires et hyperproliferatifs en utilisant des preparations topiques d'antagonistes de serotonine

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Title
See references of WO9204015A2 *

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JPH06500775A (ja) 1994-01-27
WO1992004015A2 (fr) 1992-03-19
WO1992004014A2 (fr) 1992-03-19
EP0555231A1 (fr) 1993-08-18
AU8848291A (en) 1992-03-30
CA2090688A1 (fr) 1992-03-05
WO1992004014A3 (fr) 1992-05-14
AU8499891A (en) 1992-03-30
CA2090689A1 (fr) 1992-03-05
JPH06503816A (ja) 1994-04-28
WO1992004015A3 (fr) 1992-04-16

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