JPH06503816A - Regulation of T cell proliferation through a novel 5HT1a receptor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Regulation of T cell proliferation through a novel 5HT1a receptor This application was filed on September 4, 1990. This is a continuation-in-part of filed U.S. Patent Application No. 0,715,78710. For clarification Therefore, a 5HT2-like receptor has been selected as the 5HT1a receptor.

A novel 5HT2-like receptor is present on transformed Jurkat cells. and that the receptor is present on human T lymphocytes (activated T cells). Initial research has shown that Furthermore, through experiments, 5H The presence of T receptors on activated but not resting T cells, these 5 HT receptors and 5HT receptors on gel-cut cells have similar pharmacological properties Furthermore, the 5HT receptor can induce adenylate cyclase activity under certain conditions. regulates the proliferation of CD4+ cells and promotes the proliferation of CD8+ T cells. It has been confirmed that this is possible. In addition, pharmacological, biochemical and molecular 5HT on Jurkat cells and activated T cells by a combination of structural analysis Characterization of specific receptors confirmed the existence of 5HT receptors. In addition, the receptors on Jurkat cells and activated T cells are 5H receptors. It is known to belong to the Tla tribe. In other words, the Jurcasoto cells and and activated T cells, this means that 5HTb study of signal transduction, and study of specific hominid 5HT1a origin. Evaluation of northern analysis methods using oligonucleotide probes, etc. Based on established standards. On the other hand, resting T cells are 5HT according to these criteria. b Their findings identify compounds that interact with these receptors and cell proliferation mechanisms. This has served as the basis for developing various strategies for achieving this goal.

The present invention aims to increase cell growth based on the recognition of novel serotonin receptors that exist as cell surface molecules. Concerning the regulation of reproduction. The new receptor is a serotonin receptor belonging to the 5HT2 family. Ru.

In addition, the 5HT2-like receptor is used for malignant cells or CD4+, CD8+, etc. It is present on tumor cells and even activated T cells.

The invention is also based on the recognition that proliferating cells contain/express serotonin. related to the regulation of cell proliferation.

In addition, the proliferation of malignant or tumor cells and activated T cells containing the serotonin is It can be reduced by inhibiting serotonin synthesis.

The invention also provides an effective amount of an agonist to promote or inhibit cell proliferation. The new 5HT2 receptor can be stimulated by introducing a receptor or an antagonist. Cell baths shown - Concerning the regulation of proliferation.

Many neurotransmitters bind to or exert certain effects on cells of the immune system. A substance that achieves this goal has been disclosed (see Reference 1). One such neurotransmitter is Serotonin (5-hydroxytryptamine, 5-HT) is also found at sites of inflammation. It is also the main product of platelets released (2-4). In addition, serotonin is an IFN-induced IFN induction on macrophages to enhance induced phycytosis (5) one to suppress Ia expression (6), and another to increase NK cell cytotoxicity. (7), also for influencing ion permeability in isolated Brae B lymphocytes. (8) and inhibits mitogen-promoted T cell proliferation in vitro. (9) is known. Furthermore, the serotonin and serotonin receptor condition has been shown to be required for the expression of delayed-type hypersensitivity in mouse models. It has been suggested (10-11).

In addition, the functional, pharmacological, and molecular structural properties of the serotonin receptor have been investigated in the peripheral and and the central nervous system (12). Furthermore, different works of 5HT A distinct category of receptors that mediate this function has been defined (13). Also, 5H Experiments using T antagonists shift delayed-type hypersensitivity responses to bona fide receptors. Suggestions that T lymphocytes capable of metastasizing can express 5HT receptors (11) Also, ketanserin and retanse Certain 5HT2 antagonists, such as ritanserin, enhance this response. Prevent metastasis. The purpose of the experiment reported here was to determine whether the parallel type of 5HT receptor is Can the 5HT receptor be identified on human T lymphocytes and whether the 5HT receptor is present in the nervous system? 5 Signal transducers similar to those previously reported for HT receptors The objective was to determine whether the transaction could be mediated. As a result, the hominid 7'IJn The PA system (Julkanoto) is responsible for phosphatidylinositol hydrolysis and intracellular was found to express a 5HT2-like receptor subtype that can mediate Ca+ mobilization. Ta.

In addition, the activation of body-stopping T lymphocytes may cause these cells to undergo regulation and It is critical for most immune responses to exert its activity.

Also, during this activation, cells in a relatively inactive state undergo complex processes including cell differentiation and proliferation. make a change. Furthermore, the activation of the T lymphocytes is caused by the interaction between the T cells and antigen-present cells. It is the result of ligand-receptor interactions that occur at the interface. In addition, the T-li A large number of different cell surface molecules on lymphocytes and antigen-presenting cells play a role in antigen presentation. may be involved in the above-mentioned complex cell-cell interactions that occur during cell-cell interactions. Therefore, antigen-induced T lymphocyte activity must include stimulation of the T cell antigen receptor. Ru. However, stimulation of the T cell antigen receptor alone is insufficient to induce a proliferative response. other cell surface molecules expressed on the T cell function as accessory molecules. . The auxiliary molecule can function as a conjugation molecule and initiates via the antigen receptor. modified transmembrane signals and/or their own transmembrane signals. Initiates membrane signal action.

Many of these cell surface molecules play a role in the activation, differentiation, and proliferation of T cells. appears on the surface of the T cell. Furthermore, the proliferation of T cells depends on the action of IL-2. is thought to be regulated primarily on its specific cell surface receptors. In addition , the IL-2 also plays a role in the so-called autocrine (a) that occurs in the proliferation of other T cells. It has utocrine and paracrine effects.

Furthermore, progeny cell proliferation under the action of IL-2 is the main mechanism for the T cell growth. However, under certain circumstances, the proliferation of these T cells may Occurs independently of IL-2.

It is known that a novel 5HT2-like receptor exists on the cell surface of activated T@ cells. However, the novel 5HT2-like receptor is regulated by inhibiting serotonin synthesis. It has already been recognized that it is possible to

The present invention also provides novel 5HT2 antagonists for promoting or suppressing cell proliferation. It relates to regulating the proliferation of cells that exhibit symptoms.

Furthermore, the present invention relates to the regulation of cell proliferation presenting novel 5HT2-like receptors. . The antibody has specific single or multiple epitopes for 5HT2-like receptors. Contains multiple "types" of antibodies with Furthermore, the “type” of the antibody includes polyclonal null antibodies, monoclonal antibodies, chimeric antibodies, humanized or It also includes hominid antibodies.

Furthermore, the present invention provides 5HT2-like receptors or antibodies against the 5HT2-like receptors. can produce mimotopes and small peptide ligands that bind to the body. and the regulation of cell proliferation presenting a novel 5HT2-like receptor. In addition , the small peptide ligand functions as an agonist and/or antagonist. do.

Normal diploid cell proliferation generally relies on a continuous supply of endogenous or exogenous growth factors. requires the existence of Additionally, tumor cell proliferation generally requires the addition of exogenous growth factors. It is recognized that it is not necessary. This is because tumor cells produce their own growth factors. growth factor receptors change and activate them sequentially Or, the signal transduction components may be altered to cause the tumor cells to This is caused by continuous activation. The tumor cells also have unique Unique hormones and growth factors that can express the physical condition and are not normally linked to the parent cell tissue can also be generated. Furthermore, recently, normal diploid mouse fibroblasts Expression of 5HT1c or 5HT2 receptors in cells (on gene transfection) It has been shown that a transformed phenotype can be obtained in these cells by It's turning into a crab. That is, cDNAs encoding these receptors are introduced. 5HT, which transforms the transformed cells with the secondary messenger signal. expresses high affinity receptors for. In addition, these fibroblasts Assemblage points in tissue culture are formed, allowing growth in soft agar, Tumor formation occurs in naked mice. This suggests that serotonin synthesis and 5H Variant expression of T receptors may play an important role in tumorigenesis It is. In other words, normal cells are susceptible to transforming secondary Messenger pathways. to obtain the ability to synthesize serotonin and/or serotonin receptors. From this, the transformed phenotype can be obtained. Therefore, the conclusion presented here is (1) proliferating cells contain serotonin, (2) inhibition of serotonin synthesis is a positive inhibiting cell proliferation by tumor cells rather than by normal cells; (3) normal cells; Growth of tumor cells, but not cells, is stimulated by certain serotonin receptor antagonists. It's hindered.

Mengoka Al Functionally reducing the amount of serotonin available for binding to 5HT2-like receptors or functionally reducing the effectiveness of the 5HT2-like receptor binding site. A method that suppresses the proliferation of cells exhibiting 5H1'2-like receptors consisting of Law. The method applies to malignant or tumor cells and activated T cells. The activity Sexualized T cells may be IL-2 dependent or IL-2 independent. stomach. The proliferation of these cells depends on the enzyme tryptophan hydroxyl, which inhibits serotonin synthesis. This can be inhibited by administering a sufficient amount of a compound that inhibits the activity of sylase. Current An example of such a compound is p-chlorophenylalanine.

5HT2 consisting of functionally reducing the effectiveness of 5HT2-like receptor binding sites. In a method for suppressively regulating the proliferation of cells exhibiting similar receptors, the proliferation is , an amount sufficient to bind to the 5HT2-like receptor to inhibit cell proliferation. characterized by being suppressed by introducing at least one kind of antagonist How to do it.

The at least one antagonist is selected from the group of 5HT receptor ligands. Ru. Furthermore, the group of 5HT receptor ligands includes ritanserin (ritanserin). erin), mesulergine, mianserin (mian) serin), spiperone, and the 5HT2 analogues Includes mimotopes and antibodies directed against the receptor.

5H consisting of functionally increasing the effectiveness of 5I (T2-like receptor binding sites) A method of promotingly modulating cells displaying T2-like receptors. The proliferation of said cells is introducing at least one agonist in an amount sufficient to promote cell proliferation; Therefore, it is enhanced. The at least one agonist belongs to the group of 5HT receptor ligands. selected from.

Furthermore, the group of 5HT receptor ligands includes pelanserin. , ketanserin, methylserotonin, 8-OH-D 　P　A　T.

and propranolol.

Functionally reducing the amount of serotonin available for binding to 5HT2-like receptors or functionally reducing the effectiveness of the 5HT2-like receptor binding site. It consists of inhibiting the proliferation of cells exhibiting 5HT2-like receptors. A method for treating T cell-dependent pathologies in mammals. The proliferation of these cells requires serotonin synthesis. Effective amount of a compound that inhibits the activity of the inhibiting enzyme tryptophan hydroxylase can be suppressed by administering Examples of such compounds include p-chlorophenylene Lualanine is mentioned.

5HT2s by functionally reducing the effectiveness of 5HT2-like receptor binding sites. Mammalian T cells consisting of suppressive regulation of the proliferation of cells presenting similar receptors In the method for treating a dependent disease state, the cell proliferation is inhibited by the cell proliferation. at least one antagonist in an amount sufficient to bind to the 5HT2-like receptor; A method characterized by being suppressed by introducing. the at least one type of The antagonist is selected from the group of 5HT receptor ligands. Furthermore, the 5H The group of T receptor ligands includes ritanserin, methradi mesulergin, pi renpe rone , spiperone (spiper.

ne), and mimotopes and antibodies against the 5HT2-like receptor. It will be done.

Functionally reducing the amount of serotonin available for binding to 5HT2-like receptors or functionally reduces the effectiveness of the 5HT2-like receptor binding site. Is it possible to suppress the proliferation of cells exhibiting 5HT2-like receptors? A method of treating neoplastic conditions in mammals consisting of:

The proliferation of these cells depends on the enzyme tryptophan hydroxyl, which inhibits serotonin synthesis. by administering an effective amount of a compound that inhibits the activity of the enzyme. concerned An example of a compound is p-chlorophenylalanine.

5HT2s by functionally reducing the effectiveness of 5HT2-like receptor binding sites. Mammalian neoplastic activity consisting of suppressive regulation of proliferation of cells displaying similar receptors In a method of treating a medical condition, the proliferation is inhibited by the 5H introducing an effective amount of at least one antagonist to bind to a T2-like receptor; A method characterized by being suppressed by entering. Even without making any concessions, it is a kind of a The antagonist is selected from the group of 5HT receptor ligands. Furthermore, the 5HT The group of receptor ligands includes ritanserin, mesladine (mesulergin), pirenpe rone, su spiperone and the mimetic for the 5HT2-like receptor. Includes motopes and antibodies.

5HT2 consists of functionally increasing the effectiveness of 5H-T2 analog receptor binding sites. Mammalian immunity by promoting the proliferation of T cells displaying similar receptors How to treat deficiency conditions. Proliferation of said cells is controlled by a small amount sufficient to promote said cell proliferation. Enhanced by the introduction of at least one type of agonist. At least one concession The species agonist is selected from the group of 5HT receptor ligands. Furthermore, the 5HT The group of receptor ligands includes petanserin, ketanserin ( ketanserin), methylserotonin, 8-0H-DPAT, proprano Contains roll and mianserin.

Nonaz [+ FIG. 1 is a graph showing 5HT binding to Jurkasoto cells. Applicable Jurkat cells (1xlO6) in the absence of 50 μM 5HT (non-specific binding) Incubated with indicated concentrations of 5HT in the presence (total binding) or in the presence of 5HT for 6 min at 4°C (''H). . The specific combination ([]) is expressed as the difference between these two values. Figure 1A is Show the results shown on the Scatchard plot. vinegar.

Figure 2 shows the rate of association and dissociation of (“H)5HT to Jurkat cells. This is a diagram. (A) Time course of association of (“H)5HT to Jurkat cells. to show the The conditions are the same as in "Materials and Methods" except that the culture time is different (b). This is what is described in the column.

(B) shows the time course of dissociation of (“H)5HT binding (b) from Jurkat cells. show.

Figure 3 shows the intracellular Ca''+ ion concentration in Jurkat cells at 0KT. FIG. 3 is a diagram showing a comparison of the effects of 3 and 5HT. Approximately 5 seconds after starting data collection Afterwards, 0KT3 (lμg/ml) or 5HT (5μM) was added directly to the cells. The data were collected over a total of 4 minutes. Then, the relative intracellular calcium concentration Violet fluorescence (calcium free indo-1 (indo-1)) and blue fluorescence (calcium free indo-1) Calculated from the contrast with sium release ind-1) and plotted as a function of time. .

Figure 4 shows the concentration dependence of 5HT-mediated increase in intracellular Ca'+. . Jurkat cells loaded with Indo-1 were cultured at 37°C for 2 minutes in the presence of 5HT. did.

Cells with increased intracellular Ca (■) and total intracellular Ca2+ concentration (b) The ratio of s) by measuring the increase in fluorescence. In this case, the positive cells contain 375-425 nM Ca''+, negative cells contain 150-175 nM It was assumed that the term "Ca'" was included.

Figure 5 shows 5HT-mediated increase in phosphatidylinositol turnover. FIG. -H) Labeling Jurkat cells for 48 hours in the presence of inositol Processed. Next, the labeled cells were treated with 3BM of 5HT (b) or 1Hg/ Culture in the presence of ml of 0KT3 (■) for a specified period of time, then collect the bacteria and collect the anion. IP level analysis was performed by exchange chromatography.

Figure 6A shows specific binding of 3H-5HT to T cell blasts. be.

FIG. 6B shows the binding of ketanserin to T cell embryos.

Figures 6A-1 and 6B-1 are obtained from Figures 6A and 6B, respectively. Scatchard analysis of the combined data is shown.

FIG. 7 shows a competition experiment using various 5HT receptor ligands.

FIG. 8 is a diagram showing the identification of 5HT in PBL.

FIG. 9 is a diagram showing the effect of 5HT on proliferation of PBL.

Figure 10 shows analogs that bind to 5HT receptors and promote or suppress T cell proliferation. This is a diagram.

Figure 11 shows that activation of CDS+ suppressor T cells by PWM requires 5HT. FIG.

Figure 12 shows that 0KT3 was used in the presence or absence of an analog that binds to 5HT. It is a figure showing the effect of stimulated PBL.

Figures 13A and 13B are diagrams showing the characteristics of serotonin receptors on tumor cells. Ru. (A) Jurkat cells were collected from the logarithmic growth phase, 3H-5HT (◆), 1251-LSD (◇), 3H-ketanserin (■), 3H-80H-1)P AT (b) L again! 50 M in the presence of 3H-DOB (B) (NEN) of RPM 11640 medium in the presence or absence of HT for 6 minutes at 0 to 4°C. Cultures were grown at 3×10′/ml in a total volume of 600 μm. During the relevant period (see diagram) Binding equilibrium was reached at 1). The cells were then coated with a nitrocellulose membrane (millibore (M PB84m (illipore)) and cooled the test tube and filter. The cells were washed three times each with l. Ascorbate (100 μM) was added to the washing solution. In addition, non-specific coupling to the filter was reduced (15). As a result, all specific bonds Expressed as the difference between binding and the binding observed in the presence of 50HM5HT.

Figure 13A-1 is a screenshot of combined data using 5HT (b) and ketanserin (◆). Demonstrate catchard analysis. Kd was 130 nM.

(B) Cells were cultured with 100 μnM 38-5HT and the indicated concentration of 5HTC) encerine (b), a-methylserotonin (◆), mesladine (■), 80H-D O℃ in the presence of 5HT receptor ligands such as PAT (◇) and ritanserin (b) and bound 3H-5HT was determined as described above. After that, 50HM Specific binding was determined in the presence of 5HT.

Figure 14 shows ritanserin-mediated tumor cell proliferation by 5HT receptor ligands. FIG. 3 is a diagram showing the reverse reaction of inhibition.

ME180 cells were cultured at specified concentrations in the presence or absence of ritanserin (20HM). degree 5HT ([]), perancerin (◆), ketanserin (b), mianseri 5HT receptors such as (◇), 80H-DPAT (■), propranolol (b), etc. The cells were cultured for 48 hours in the presence or absence of body ligand. Then 1 μCi” Labeling was performed during the last 6 hours of the culture using H-TdR and then harvested. The incorporation into DNA was measured. Results of ritanserin or other ligands As the rate of control of H-TdR uptake into ME180 cell culture in the absence of It was expressed as 3H-TdR uptake by ME180 cells occurs in the absence of ritanserin. 40679+/-2548 in the presence of ritanserin and 2215+/-2548 in the presence of ritanserin. It was +/-433. Therefore, these ligands have their specific 5HT receptors. It acts on the body part between 11-1 On.

Figure 15 shows 5HT-dependent changes in intracellular cAMP content in T cell embryos. be.

Figure 16 shows cAMP content (0) and proliferation in T cell culture after addition of 5HT. It is a figure showing the rate of change of (.).

Figure 17 is a diagram showing the inhibition of the antiproliferative effect on gamma interferon (IFN). be. (A) ME180 cells were treated with 300 μM tryptophan (■) or 5 μM 5 The cells were cultured for 3 days using IFN in the presence or absence (b) of Htp (♦).

(B) ME180 cells were treated with different concentrations of 5Htp ([]) or tryptophore. Cultured for 3 days using 100 u/ml IFN in the presence of fan (◆) . Thereafter, after treatment with 1 μCi of “H-TdR for 4 hours, cell proliferation in the control “H-TdR was 88,765±1,256 cpm.

Figure 18 shows a comparison of the ability of 5Htp to reverse the antiproliferative effect of gamma interferon. FIG. 5 shows the metabolites of 58tp for ME180 cells were treated with 5H at a specified concentration. tp (○), 5HT (・), melatonin (■), N-methylserotonin (Ro) ) or 100 in the presence or absence of 5-hydroxyindoleacetic acid (×) Cultured for 3 days using u/ml IFN. Control growth was 73456±2289 cpm, and 16894±1145 when grown in the presence of IFN.

Figure 19 shows cell loss of tryptophan and 5HT after incubation with gamma IFN. FIG. ME180 cells were incubated in the presence of 100 u/ml IFN (○) or After a predetermined number of days have passed in the absence of (○), it is collected and 5HT The content (upper figure) and tryptophan content (lower figure) were analyzed, respectively.

Figure 20 shows that 5Htp is extracellular tryptophore in culture using gamma IFN. FIG. M E 180 cells 100u/ ml of 10HM5Htp in the presence (·, oral) or absence (0) of IFN. Cultured in the presence (・) or absence (b).

The culture medium was collected after a specified number of days, and the tryptophan content was determined by HPLC. analyzed.

Figure 21 shows that 5Htp reverses the inhibition of cell proliferation by low tryptophan. FIG. ME180 cells were cultured in RPMI-1640 medium with a predetermined amount of aliquots. using putophane in the presence (·) or absence (○) of 3 μM 58tp. Cultured for 48 hours. The results are expressed as “H-TdR uptake” after 6 hours of treatment on the second day. It was shown.

B-3 short days First, we need to determine whether 5HT receptor subtypes can be identified on human T lymphocytes and whether 5HT receptor subtypes can be identified on human T lymphocytes. A study was conducted to determine whether the condition could mediate signal transduction. Ta.

Here, 5HT indicates serotonin or 5-hydroxytibutamine, and DOB is (R)-(-)-4-bromo-2,5-dimethoxy-amphetamine, 80 H-DPAT is 8-hydroxy-2-(di-n-propylamine) tetralin, IP is inositol phosphate, IC50 is the concentration required to inhibit the response by 50%. In addition, EC50 is the concentration required to produce half the maximum response! H-5h t (20Ci/mmol), “H-ketanserin (65Ci/mmol) "'-1 lysergic acid diamide (LSD, 2200Ci/mmol) , “H-80H-DPAT (128Ci/mmol) and 3H-myo -Inositol (48Ci/mmo I) was added to New England Nuclear ( New Eng I and Nuclear). Fe2. RPM I1640 and L-glutamine were obtained from GIBCO. Also , India-1 to Sigma Chemical Co., Ltd. ).

■ Jurkat cells were collected from the American Type Culture Collection (American Obtained from Type CultureCo I 1ect 1on) and 10% RPM11640 in the absence of antibody and in the presence of 1mM glutamine with the addition of FC3. Displace 1-21 cultures of Jurkat cells maintained in continuous culture in medium. Collected by heart isolation, containing 1mM EDTA, pI-(7,5(7)I-Lis hydrochloride) Suspend in 10mM solution and incubate on ice for 10 minutes, then use a homogenizer (Dou homogenizer) to make the total amount 2020-4O.

The homogenate was then centrifuged at 110,000x for 10 minutes to separate the nuclei and cells. removed. The supernatant was further centrifuged at 1,100,000x for 1 hour.

Next, the pellet was suspended in EDTA-containing Tris-HCl buffer to give 7.7 mg. The protein concentration was set at 1/ml. The sample membrane was kept at -70°C until used for binding assays. saved.

Paste project ±4 Joule-cut cells were harvested from a culture in the logarithmic growth phase, and the total volume was 600 μm. xlO'/ml or at 0 to 4°C in the presence of RPM11640. 5HT agonist or antagonist in the presence of HT (see text for details) Incubation was carried out for 6 minutes in the presence or absence. Binding equilibrium was reached during the culture. Ma In addition, a substitution reaction was performed by culturing the cells with 3H-5HT, and then the text Culture with unlabeled agonist or antagonist as described above. It was. The cells are then collected on a glass fiber filter, the test tube and the filter The container was washed three times with 34 ml of chilled PB. Next, “5I-LSD or” Ascorbate (100 μM) at the time H-80H-DPAT binding was assessed. was added to the wash solution to reduce non-specific binding to the filter (14-15).

The cleaning time for each filter is 20 seconds or less. Also, 'H-5HT Juru cut The typical value of total binding to cells is 10100n total added cpm number: 10') of "H- It was 110,000-15,000 cp in the presence of 5HT. In addition, 100 μM 5HT (non-specific binding), approximately 1500-3000 cpm remains bound. It was. Next, to examine the association rate, cells were incubated with 100 nM 3H-5. Cultured in the presence and absence of 100 μM 5HT for various times. . The binding reaction was then stopped by the addition of PBS and filtered. Similarly, the dissociation rate To examine the temperature, cells were incubated at 0 to 4°C in the presence of 1100 n of 5H-5HT. The cells were incubated for 10 minutes. Then, 5HT was added to a final concentration of 100 μM. Then, the reaction was stopped by adding cold PBS and the sample was filtered. like this Binding assays were performed in duplicate a minimum of three times, with the duplicate samples 5% of each other. It was within

Binding assays using sample membranes of Jurkat cells were performed with total protein content ranging from 500 to 70%. Similar procedures were carried out in Tris-HCl buffer, pH 7.5 at 0 μg/ml. I followed the steps.

1 scallop top 11 A device with improved intracellular Ca + mobilization (Bect.

n Dickinson FAC8TARPlus) using the India-1 method. It was evaluated as follows (16-17).

Briefly, Jurkat cells were incubated with DME in the presence of 10 mM HEPES for 45 minutes at 37°C. cultured with 2 μM Indo-1 in M and then washed 3 times to 106 cells/ml. and suspended in PBS. A portion (1 ml) of the modified sample was kept at 37°C. I held it in place and started collecting data. Approximately 5 seconds after starting data collection then add 5HT or 0KT3 (see text for concentration) to the sample. Direct addition and data acquisition over a total of 4 minutes. Fluorescent light is emitted by a condensing lens split by a long optical path filter (455nm cutoff) placed at 45° to and passes through two bandpass filters BP485/22 and BP395/20. measured at the same time. The set of collected data is then processed to produce violet fluorescence (39 5/20 nm) to blue fluorescence (485/22 nm) as a function of time. and get it. By doing this, the calcium concentration can be calculated using the following formula. Ru.

[Ca”+ko=Kd・(R-Rm i n)/(Rma x-R)・ S f 2/S b 2 In the above formula, Kd is the dissociation constant (250 nm), R, Rmin and Rm ax is the respective fluorescence intensity at resting state, zero and saturated calcium concentration Sf2/Sb2 is the value of the ratio, Sf2/Sb2 is the ratio of calcium-free and calcium-binding dyes. Ratio values of fluorescence intensity are shown (18).

We also investigated the response of individual Jurkat cells to total population. ) The influence of the concentration of 4T was evaluated.

In this case, the cells were kept at 37°C, and 5) FT was added and incubated for 2 minutes. After that, 1 0000 data lists were collected. The data is then divided into violet fluorescence and blue fluorescence. The ratio of the number of cells corresponding to approximately twice the standard deviation threshold was determined. this The experiment was conducted a minimum of three times and similar results were obtained.

Phosphorus inositol level Jurkat cells (2x 10'/ml) were treated with 10 μCi/ml of 3H-Myo. - 1% dialysis in RPM II 640 medium using inositol in the presence of FC3 for 48 hours Labeled in between. Thereafter, cells were harvested and placed in RPMII640 medium without Fe2. Washed twice with The cells were then incubated in medium without FC3 at 37°C, lXl0'/ ml and treated with <5HT as described in the text, followed by centrifugation several times. collected. The extract was then neutralized with 7N KOH and anionized as described above. IP level values were analyzed by exchange chromatography (19). Create double The level of inositol phosphate was measured three times. and obtained similar results.

PB Wako Momme 11 PBMC were obtained from blood buffy coats obtained from normal donors. Next, the current The PBMCs were subjected to lymph isolation gradients (Ph armacia, Piscataway, NJ)) and diluted in PBS. Washed twice. After that, 10% DMSO-90% FC3 (GIBCO, Grand 15land, NY) and stored in liquid nitrogen until use. . Cell surface Ag and proliferation responses of PBMCs after storage in liquid nitrogen were compared with fresh The lymphocyte characteristics were identical.

PWM and PHA from GIBCO (OKT3 from mouse (BALB/c m1ce) ) and used at a dilution of 1:1000) and diluted 1/80 in PBS. It was diluted and used for culture. Additionally, purified recombinant or natural IL-2 can be Boehringer Mannheim (Indiana) polis.

Purchased from IN)). 1 unit of I L-2 activity in a 24 hour assay Determined as the amount required to cause 50% maximal proliferation of the L-2 dependent cell line CTLL. I justified it.

In addition, purified rIL-1, rIL-4, and rIL-6 were purified using Genzyme (Genzyme). Zyme (Boston, MA).

1 Itching I PBMC, CD4+ cells MC or CD4+ cells with 10% FC3 added without antibody flat-bottom 96-well microorganisms for 3 to 8 days in Canadian RPMI-1640 medium. Cloculture plate (Becton Dickins.

n) in a humidified atmosphere of 5% CO in air to a concentration of 5 x 10' cells/ml. The cells were cultured in a total volume of 100 to 200 μl. Then, uptake of ('H)TdR (8H)thymidine (2Ci/mmol, Amer sham International, Indianapolis, IN) , the cells are filter paper (PHD cell harvester, Chambr idge, MA) in duplicate, 18 to 20 hours before harvesting. Added. The data represent the average uptake of ('H)TdR in the duplicate sample. Shows the average value. In addition, the number of duplicate samples is within 5%, and We have conducted these experiments at least three times with similar results, and we will use them in the next experiment. This was reproduced as a positive control.

Application to microbial assay: 16:1706° The same procedure as described on pages 15-16 was performed for cells.

Table 11 5. Inhibition of HT synthesis results in inhibition of proliferation by tumor cell lines. ME180 cervical canal Carcinoma cells were obtained from ATCC and maintained as described in (). Then, the cells were cultured for 48 hours, and cell proliferation was determined by either 5HT content or the addition of H-TdR. We conducted an analysis on this. 3H-TdR: Separate the medium and cells and add 5HT. After extraction, proteins were precipitated with 70% ethanol. Then transfer the sample to the device ( It was concentrated using SP e d-v a c, ).

The 5HT content of the sample was then determined using a 280 nm excitation filter and a 360 nm emission filter. HPLC (Hew The analysis was carried out by L e t-Packard). A (pH 3.0 with phosphoric acid) (50mM) ethylamine) and B (adjusted to pH 3.0 with 40% methanol) Two mobile phases were prepared consisting of 50mM)-ethylamine). The slope is A1 00% to B100% in 25 minutes (51). Standard retention time I did it like this: 5 HT.

3.50 minutes, 50■4-tryptophan, 4.77 minutes, tryptophan, 9.1 1 min, 50H-indoleacetic acid, 12.5 min, and melatonin, 21.69 Minutes. Again, cells were treated with 1 μCi of 3H-T d during the last 4 hours of culture. The cells were treated with R and collected to measure their incorporation into DNA. Then, the culture was carried out for three times. It went seriously. Actual 5HT synthesis from tryptophan with 3-H tryptophan Confirmed by culturing cells, extracting soluble components, and extracting methylpropanol. Solvent system of ethyl acetate-ammonium hydroxide-water (45-30-17-8) 3H-5HT was analyzed by thin layer chromatography on a TEM (52). this Under these conditions, the Rf value of tryptophan is i;to, 03, and 5HT The Rf value was 0°50.

As a model system, “H- 5HT binding was investigated to determine whether T cells express specific binding sites for the neurotransmitter. I decided to do it. These cells respond to signal triggers stimulated by antigens and mitogens. Previously used by many researchers to explore the mechanism of transduction (20-21). The results shown in the first figure indicate "H-" for Jurkat cells. Binding of 5HT is detectable, indicating that the binding can be saturated. Also , the label was determined from the amount of H-5HT binding in the presence of 5HT at a given concentration (Figure 1). Difference in the amount of H-5HT binding in the presence of 100 μM of uncoupled 5HT Specific bonds were determined by subtraction. In addition, these 58T concentrations Therefore, the specific binding is about 70-80% of the total binding, and the maximum binding of 3H-5HT is The ratio is reached within 5 minutes of incubation (Figure 2). In addition, the bond of 5HT is 1 to Maximum at 2 μM 5HT. Figure 1A is the scanocha of the combined data. It shows a code plot. Additionally, data from three separate experiments provide a solution. An average value of separation constant (Kd) of 90 nM was obtained. In addition, 'H-5HT's Jurukasoto The binding site density for cells is 130 fmol per 10 cells or 1 cell. There were 5oooo receptors per cell. In addition, Hillthro The Hi l l s 1 ope value was 0.8 (not shown). moreover , various binding conditions to investigate the effects on affinity constants and receptor density. It was investigated. As a result, Mg++ ions up to 2mM, balloon ions up to 10μM EDTA up to 3mM or imibramine up to 10μM (5HT capture It was found that the addition of (blocking (not shown). Please note that all results shown here are derived from the logarithmic growth phase. . In addition, Jurkat cells obtained from cultures exceeding 10' cells/ml are 5HT-receptive. There are practically fewer bodies. Furthermore, these cultures also transferred Ca++ ions. No response to 5HT by increasing the concentration of inositol phosphate .

Isolated membrane samples also confer similar properties to 5HT.

Furthermore, 5HT binding reaches saturation at 1-2 μM 5HT, leading to scattering. The average value of Kd determined by Chard analysis was 160 nM. Also, the relevant The rates of 5HT binding and dissociation (see below) are similar to those for intact cells and membrane samples. It was like that. Furthermore, similar concentrations of 5HT or 5HT agonists or antagonists Concentrations of antagonist prevent binding of H-5HT to membrane samples or intact cells. be done. Additionally, the main difference between binding of 5HT to sample membranes and binding to intact cells is This is revealed by calculating the number of bodies/cells. As a result, in a complete cell, the cell light The number of receptors per membrane is 5oooo, and the number in isolated membrane samples is 300. It was 00. At present, it is unclear what causes this difference. deer The binding data showed that the intact cell biological response to 5HT as a function of 5HT concentration Since only the results of 5HT binding to the intact cells are contrasted with the responses, I will report it here.

The rate of binding was then examined. From the results in Figure 2a, we can see that “H-5HT binding” It can be seen that this was completed in 4 minutes, with half of the maximum binding occurring in 30 seconds. Joining takes 10 minutes It was maintained for a while. From these data, 2. lXl0IO/m.

The association rate constant (kl) of 1-m1n can be calculated based on the following formula.

k1= [1/l (a-b) In [b (a-x) / a (b-X)] , In the equation, a is the initial concentration of H-5HT, b is the receptor concentration, and X is the time is the amount of receptor-ligand concentration at time t. Furthermore, the receptor-ligand complex The coalescence dissociated rapidly in the presence of excess unlabeled 5HT. These results is shown in Figure 2b. The pseudo-order dissociation rate constant is 11/min and can be calculated using the following formula: Wear.

k, 2 = (1/l) I n (a/x), where a is the bond at time t is the amount of ``H-5HT'' (15).Furthermore, the value of the ratio of ks divided by 1 is is the calculated value of Kd, which is 180 nM. This value is theoretically Scatchard It agrees well with the Kd value calculated by analysis.

Furthermore, the cell surface receptors for 5HT are characterized by pharmacological and functional properties. 5HT1a or 5H of 5HT, which is classified as a few ordered types (12-13) Binding to the T1b site inhibits stimulated adenylate cyclase activity and 5HT Binding to the 1c or 5HT2 site is determined by the level of inositol phosphate and the intracellular Increase Ca++a degree. At this time, Jurkat cells were converted into Indo-1 cells. Incubate with fluorescent dyes that are sensitive to changes in Ca + + 5 μM of 58T or 1a. After culturing in the presence or absence of g/ml 0KT3 as described in the Methods Analyzed by flow cytometry. These results are shown in FIG. Ma Cells cultured in PBS were incubated with 1l50-175n of Haeslein calcium concentration. The intracellular Ca+ concentration did not change during 10 minutes. Also, In Julcant cells cultured in the presence of 0KT3, the intracellular Ca+ concentration increased within 2 minutes. It increased to a maximum of 2 μM within a period of time and then decreased to a level of 700 nM. difference Furthermore, in Julcasoto cells cultured in the presence of 5 μM 5) IT, the intracellular Ca +10 concentration increased to 400 nM within 2 minutes. In addition, the 5HT-treated Jurkat cells are characteristic of the Jurkasot cell response to 0KT3. , did not show a large initial peak in [Ca++] (20-21). Fourth The figure shows the increase in intracellular Ca++ concentration and 5HT concentration in the Jurkat cell culture. shows a comparison with the Ca++ concentration in individual Jurkasoto cells as a function of There is.

In this case, at a given 58T concentration, the cells have a low Ca+ concentration (150-17 5nM) and one with high Ca+ concentration (375-425nM). It can be divided into. Therefore, the response of the entire culture shown in Figure 3 is that the low intracellular Average of individual cells with either Ca+ tens or high intracellular Ca++ concentrations It can be expressed practically as Note that the increase in the intracellular Ca + This means an increase in cells with a higher intracellular Ca+ concentration. Additionally, half of the maximum response was between 100 and 300 nM 5HT, which was obtained from the binding data above. Almost comparable to the dissociation constant.

Increased intracellular Ca+ concentration is due to phosphatidylinositol biophosphorus concentration. can be mediated by IP3 produced by hydrolysis of ate (22).

Binding of 5HT to the 5HT2 receptor family induces inositol phosphate in nervous tissue. was found to increase the level of

In this case, Jurkat T cells were labeled with 3H-inositol for 48 hours and Stimulation was performed with μM 5HT or 1 μg/ml 0KT3. The cells were then divided into 5 Samples were taken at various times after the addition of HT or 0KT3 and analyzed using anion exchange chromatography. The level of IP was analyzed by matography. From the results shown in Figure 5, the relevant I P levels increase after addition of 5HT or 0KT3 and reach maximum within 1 minute You can see that you have reached it. The IP level then decreases over a period of 10 minutes; approached the baseline level. Note that under these conditions, 5HT and 0 A similar increase in IP levels was seen in both cases of KT3.

Furthermore, many 5HT agonists or or an antagonist was used. As a result, one of the 5HT2 receptor antagonists ketanserin, which is a species, and alphamethine, a type of 5HT2 receptor agonist. Lucerotonin and 5HT inhibit the binding of H-5HT to Jurkat cells. harmed. In this case, the concentration that inhibits 50% of the specific binding of H-5HT (IC50 ) for ketanserin, alpha methyl serotonin and 5HT, respectively. The concentrations were 20HM, 3μM and 0.8μM. In addition, the specific agglutinin of 5HTb 80H-DPAT, which is a type of agonist, and a specific antagonist of 5HT3 receptor. IC3-205930, a type of anti-inflammatory drug, inhibits the effects of 3H-5HT on Jurkat cells. did not prevent binding. In addition, two other 5HT1c/2 receptor antagonists Mianserin and mesladine, which are active agents, inhibit the binding of 5HT to Jurkat cells. It didn't interfere. These results are shown in Table 1. Also, ritanserin and peran Other 5HT2 receptor antagonists, such as serine, also inhibit 5HT binding. There was no (not shown). In addition, “’I-L S DS” H-ketanserin and 'H-DOB as the site of 5HT1c or 5HT2 in the central nervous system. used to label. In addition, 'H-80HDPAT was administered to the central nervous system. was used to label the 5HT1a site. Table 2 lists these ligands. K determined by Scatchard analysis for comparison of binding to Jurkat cells. Shown together with d value.

In this case, specific binding was determined in the presence of 100 μM 5HT. As a result, recruitment Under these conditions, sH-ketanserin and ``H-5HT'' can stimulate Jurkat cells. ``I-LSD and ``H-DOB label the Jurkat cells. There wasn't. Furthermore, “H-80HDPAT also labeled Jurkat cells. There wasn't. Note that the binding constant for ketanserin was 1100n.

In addition, the identification of each of these 5HT receptor agonists and antagonists Affinity constants for receptor groups ranged from 1 to 5 HM.

Next, the Ca + 10 transfer of alpha methyl serotonin, the 5HT2 agonist, The effect of promoting motility was tested (Table 3). As a result, compared to 3μM 5HT, Therefore, 10HM alpha methyl serotonin produced the maximum Ca+ mobility. Similarly, the concentration required to produce half maximal stimulation (EC50) is 200 nM 5 Compared to HT, alpha methyl serotonin was 1 μM.

Moreover, the substitution value of the bound 5HT is the same, and furthermore, the substitution value of the bound 5HT is The IC50 of alpha methyl serotonin was higher than that of 800 nM 5HT. The concentration was 3 μM. Table 4 also shows some 5HT2 antagonists. Comparison of the inhibitory effect of 5HT-mediated Ca + mobilization in Nyurkat cells is shown. There is. Of the antagonists tested, only ketanserin was Inhibited Ca+10 movement. Moreover, this IC50 value was 10HM.

Table 1 Inhibition of 5HT binding by 5HT receptor Agonist/Antagonist Antagonist 0 type IC50”5HT endogenous 0.8μM Ketanserin 5HT2 20HM alpha methyl serotonin 5HT2 aμM 8-OH-DPAT 5HT1a>50HMIC3-205-930 5HT3>20HM Mesradine 5HT1c/2 >50HM Mianserin 5HT1c 2 >5 0 M(a) The binding treatment is carried out using 2 x 10' Jurkat cells, Presence of 100n'H-5HT and various concentrations of agonists and antagonists It was performed as described in the "Methods" section below.

Table 2 Combined 5HT'B-0 ligand in Jurkat 5HT receptor ligand Kd" Kd' 5HT part - Jurkat 2 3H-ketanserin 1100n ~ 1nM 5HT2"5I-LSD > 1O nM ~ 1nM 5HT1c3H-DOB > 1100n" 1nM 5HT2 a (a) Jurkat cells were treated with 3H-ketanserin (1-500 nM), 1 2’I-LSD (0,3-3-1On or “H-DOB” (0,5-5-2 The cells were cultured at 0-4°C for 10 minutes in the presence of 00n. Specification in the presence of 100μM 5HT The binding was determined.

(b) The stated values were obtained from references 12 and 13.

Table 3 Ca + decadal agonist 3H-5HT Ca + decadal movement by 5HT2 agonist Mobility replacement IC50° EC50b 5HT 800nM 200nM Determination of substitution of alpha methyl serotonin 3M LM(a)-3H-5HT The data for this were obtained from Figure 5.

(b) EC50 values of Jurkat fines using various concentrations of 5HT2 agonists. Determined by culturing cells and then examining changes in Ca+ concentration as a function of time. Established.

Note that the rate of change of Ca+ was linear in the first 60 seconds, and Complete within 2-3 minutes after addition of the nist.

Table 4 Inhibition of Ca + 10 mobility by 5HT2 antagonists Ca + 10 mobility Ca++ nM" IC50b- 5HT (lμM) 425-1 Ten ketanserin 285 1OnM + Perancerin 435 > 1OnM + Mianserin 440 > 1 OnM + Methradine 460, >10M (a) Before adding 5HT, Jurkat cells (IXIO'/ml) were added at 1 Incubation was carried out for 5 minutes in the presence of the designated antagonist of OnM. Next, the Julka 2 minutes after addition of 5HT, the Ca + concentration in the cells was increased as described in "Methods". as determined by flow cytometry. At present, there is no corresponding response to 5HT. The answer is still linear in time (Figure 4).

(b) IC50 value is Ca + mobilization by Jurkat cells in the presence of 1 μM 5HT. The concentration of agonist that inhibits 50% of the

The neurotransmitter 5HT is released during inflammation and contributes to delayed-type hypersensitivity responses (3,4,10) is believed to play an important role in On the other hand, ketanserin and ritanserin Antagonists of 5HT, such as phosphorus, can block the DTH response in the animal body. is shown (10-11).

Additionally, treatment of certain antigen-specific T cell clones with said 5HT antagonists inhibit their ability to transfer the DTH response to bona fide receptors. However, two A functional 5HT receptor that transduces intracellular signals via the next metsenger. have not yet been identified on the T cells.

That is, the purpose of the experiments described here was to establish that the functional receptor for 5HT is Can they be identified on T-cells and whether they exist in the central or peripheral nervous system? The biological It is an analysis of both chemistry and pharmacology.

Next, we will discuss functionally and pharmacologically the subtypes of the 5HT receptor. We also characterized the molecular structure (12-13), in this case 5HT1a, 5 5HT1 group consisting of HT1b and 5HT1d, 5HT1c.

The 5HT2 group, consisting of 5HT2a and 5HT2b, and the currently only factor Three 5HT groups were used. Also, 5HT 1a, 5HT 1c and identified cDNAs encoding 5HT2 and 5HT2 receptors, and demonstrated that the cDNAs It is a G protein-linked receptor with seven types of membrane spanning units. It was found that the protein encodes a protein (23-25). Functionally, 5HT Binding to the 5HT1a site alters the activity of adenylate cyclase26) , alters potassium ion pathways and further inhibits neuronal transmission (12-13 ).

We have also identified specific agonists that bind exclusively to the 5HT1a site. these Two of them are 80H-DPAT and Ibusavirone (ipsap irone) It is. Furthermore, whether 5HT binds to the 5HT1c or 5HT2 site or It can increase the turnover of tidylinositol and the movement of intracellular Ca+ Understood. It also has equivalent affinity with 5HT1c and 5HT2. A number of 5HT2 antagonists that interact with each other have been identified. In addition, the person concerned Examples of agonists include methazine and mianserin. Also, the other 5 HT2 antagonists include ketanserin, ritanserin and pelanceri There are many examples. These ligands are used to label the 5HT2 site, and the 5HT It has nanomole-level affinity for two sites, and also inhibits 5HT-mediated phosphatidylinonthole turnover in Suru (12-13).

Note that the 5HT2 group may additionally include a heterologous factor. Also, Phosph The 5HT site that mediates the turnover of atidylinositol is located in the hippocampal region of the brain. It was identified in the forebrain. However, in these organizations, the phosphor Tidylinositol turnover is extremely high (normal for 5HT2 site) by ketanserin with an affinity of 1-10HM to 11-1On). (27-28). On the other hand, 5HT3 receptors are mainly found in the peripheral nervous system. It looks like it is.

Note that these receptors are affected by 5HT1 and/or 5HT2 selective ligands. I don't receive it. However, certain ligands such as IC3205903 It was confirmed that the effects of 5HT can be blocked in (12-13).

In addition, the Kd value for 5HT binding to Jurkasoto cells is determined by Scatchard. 90 nM as determined by analysis and 90 nM as determined by kinetic analysis. It was 180 nM. In addition, the density of 5HT receptor sites in Jurcasoto cells 1 There were 130 Imol per 06 cells or 5oooo sites per cell.

That is, 5HT stimulates the turnover of phosphatidylinositol, Increases intracellular Ca + + in these cells. Furthermore, saturation of the 5HT bond and The largest Ca++ responses could be observed at 1-3 μM 5HT. Also, the maximum A state corresponding to half of the Ca + + response was observed at 200 nM 5HT; The value is close to the Kd value for binding of 5HT to Jurkat cells. In addition, 3H- 5HT binding and 5HT-mediated Ca+ mobilization are inhibited by specific 5HT1a agonists8. 0 H-D P AT and specific 5HT3 ligand I C5-205930. It didn't affect me. Furthermore, the 5HT and 80H-DPAT are The concentration of cAMP in the cells remained unchanged (data not shown). These data is consistent with the binding of 5HT to the above-mentioned 5HT2 receptor group. In addition, 5HT 2 agonist alpha methyl serotonin maleate inhibits bound 5HT. and stimulated Ca+ mobilization in Jurkat cells. Shigashi, Ketanse Phosphorus has a weak affinity (Kd of 100n, M) for the Jurkat cells. Jurkat of 125I-LSD (substituted by 5HT) No binding to cells was detected. Also, mianserin, methazine, pela nserin and ritanserin (all 5HT2 antagonists) at ≦20 μM concentration does not inhibit 5HT binding to Jurkat cells and also inhibits 5HT-mediated binding of 5HT to Jurkat cells. Ca++ movement was also not inhibited. However, only ketanserin placed 5HT at high concentrations. and inhibited Ca+ mobilization (50% at 10HM). Furthermore, specific 5 HT The 2a ligand 3H-DOB did not label Jurkat cells.

In addition, when considering the 5HT binding in comparison with the Ca+ mobility, the 5HT antagonist The effect of the list can be considered to be equivalent. From the above results, the gel cut 5HT receptors on cells are 5HT1c, 5) fT2 or 5HT2b receptors. It can be said that it is neither. Furthermore, it binds 5HT and phosphatidyl - mediates the turnover of inositol, but at micromolar concentrations more similar to that seen on the hippocampal region mentioned above, which is only inhibited by encerin. It looks like a sea bream (27-28). This indicates that additional heterologous factors may affect the 5HT2 group. This suggests that it exists.

The data presented here describe 5HT receptors on hominid Jurkat cells and also The receptor stimulates phosphatidyl-inositol turnover, and This shows that the intracellular Ca+ concentration in these cells is increased.

The 5HT-induced increase in inositol phosphate levels is associated with 0KT3. Similar to the increase caused by. In addition, the increase in Ca + + concentration due to 5HT The addition was from 175 nM to 400 nM within 2 minutes. On the other hand, Ca due to 0KT3 The +10 concentration increase was up to 2 μM within 2 minutes. Furthermore, this increased value is then rapidly decreased to 700 nM within an additional minute. However, these data , the biological or immunological relationship between 5HT and its receptor on the gel-cut cells. It does not represent the results of interactions. However, the above-mentioned phosphatidyl inosites Turnover of protein plays a role in signal transduction in T cell activity (see 29). stimulation of this pathway by 5HT has important immunological consequences. bring. Furthermore, 5HT is also abundant at sites of inflammation and stimulates the T cells. Function can be altered. . In addition, 5HT is a major accumulation product of platelets. It is released upon aggregation of platelets at the site of inflammation. What should be noted is that the blood The level of 5HT in the lamina is associated with diseases such as rheumatoid arthritis and systemic erythematous lupus. It is decreased by autoimmunity (30). Furthermore, 5HT2 antagonist Inflammation and association with streptococcal cell wall extracts in rats with normal resistance It has been shown to exacerbate the joint inflammation response (31). However, at present, this It is not clear that these effects are expressed through 5HT receptors on T cells.

Therefore, depending on the effectiveness of the specific 5HT antagonist or agonist, It is important to understand the immunological interaction between 5HT and 5HT receptors on sexualized T cells. and furthermore, in the in vivo immune and inflammatory response on such T cells. It is thought that the role of the 5HT receptor will become clearer. Finally, (experimentally) 5HT receptor analogues exist in human peripheral T cell cultures, but they are dormant. It is not present on stopped human peripheral T cells.

Novel 5HT2-like receptor exists on transformed cells (Jurkat cells) Following the demonstration that the receptor acts on human T lymphocytes (activated T cells), ) We conducted further experiments to prove that it exists.

T no 5 HT melon 0 5HT receptors on gel-cut cells have a “H- Weak affinity for 5HT (100nM) and ketanserin (100nM) I have a 3. Specific binding of H-5HT to T cell embryos is detectable. and saturable. That is, about 80% of the bound 3H-5HT is 10 Displaced by 0 μM 5HT, the binding was saturated with 1-3 μM 5HT. saturation Sometimes 200-250 fmol of 5HT was bound per 10 T cells. Ma In addition, 5HT binding was saturated at 1-3 μM 5HT. In this case, the Kd value is The value was 180 nM (values from three separate experiments). average). As a result, the T cell embryo has 224 fmol per 106 cells. of 5HT, with a site density of 120,000 receptors per cell. I understand that. The results are shown in Figure 6A. Figure 6A-1 shows the skim of the combined data. This shows the Catchard analysis. That is, based on the three separate experimental data, The average dissociation constant was 200 nM. In addition, Figure 6B shows the effects of T cells on embryos. The binding of tanserine is shown. The binding is saturable and the embryos of T cells have the same number expressed ketanserin and 5HT receptors. In addition, the specific bond of ketanserin was determined in the presence or absence of 100 μM 58T. No. 6B-1 The figure shows the Scatchard analysis of the ketanserin binding data, and the average solution The separation constant is 400 nM.

From the above, these data indicate that a single receptor exists on the T cell matrix, and that it is 5H. binds both T and ketanserin, respectively, in their defined concentration ranges. This is shown conceptually.

As a result of competitive experiments using various 5HT receptor ligands, we found that 5HT, alcohol H-5 for binding of phamethylserotonin and ketanserin to T cells It was found that it was comparable to HT (Fig. 7). Note that the IC50 values are 0. They were 1 HM, 0.06 μM and 20 HM. Furthermore, 80H-DPAT (5 specific for HT1a receptor), IC3-205930 (specific for 5HT3 receptor) specific for 5HT1c and 5HT2 receptors), mianserin (specific for 5HT1c and 5HT2 receptors); ) and 5HT receptor ligands such as spiberone (specific for 5HT2 receptors) was not comparable to 5HT in binding of the T cells to the embryo. Also, said 5 ) (The characteristics of the T receptor on the clay of the T cell are those of the 5HT receptor on the gel cut cell. It is comparable to the properties of receptors found in nerve tissue, and is clearly distinguishable from the properties of receptors found in nervous tissue. Wear.

The presence of the 5HT receptor is then investigated in many different T cell populations. was evaluated. In this case, the resting lymphocytes are T-cell embryos (150,000/ (24,000/cell) showed lower levels of receptors (24,000/cell). Also, C Both D4+ and CDB+ T cell lines exhibit high levels of 5HT receptors1430 00 and 171,000 receptors/cell), and the CD3+4-8- T cell line has 5 Shows a level of 3000 receptors/cell, a value observed on resting lymphocytes It is only slightly higher than what was reported. Furthermore, using different T cell mitogens, The expression of the 5HT receptor was examined as a function of time after the application of the stimulus. So As a result, the increase in receptors was linear for 72 hours after stimulation. In addition, three types of T cell togen, PHΔ, PWM, and 0KT3 increase the number of 5HT receptors on the T cells. showed no substantial difference (Table 8).

Table 8 Cell 1 liter Kosumi Kogyo Fmol/108 cells Receptor/cell PBL 40±3 24000 T-cell embryo 250±11 150,000 CD4+ T cell line 238±9 143,000 CD8+ T cell line 285± 5 171,000 lymphocytes The increase in intracellular cAMP is associated with proliferation and effecta of the lymphocytes. ctor) function (57). Conversely, an increase in intracellular Ca++ activation of lymphocytes by antigen or mitogen signals, followed by Generally recognized as necessary for lymphocyte proliferation and effector function There is. Furthermore, the results shown here indicate that it can mediate the increase in 5Hb. Ru. Therefore, this naturally limits the proliferation of lymphocytes and their effector functions. improves. In addition, the 5Hb It regulates the levels of adenylate cyclase and cAMP. That is, the ad Depending on the state of activation of nilate cyclase, 5HT1a agonists may affect cAMP levels. It can increase or suppress the lymphocytes, and also improve the proliferation and function of lymphocytes. can be suppressed or suppressed.

5HT, specific 5HT1a agonist 80HDPAT and 5HT1a antagonist Embryonic Biological Responses of Nyurkat Cells and T Cells to the Gonist Spiberone Answer Jurkat cell T cell embryo Conditions Ca++ cAMP Ca++ cAMP=--'-' (nM) (m ol) (nM) (Yu ■--2004, 72601, 8 5HT+ 610 4.9 260 0.65HT+ Subiberon 225 4, 7 250 1.480T (DPAT 470 4.8 255 0.58 0 0 D P A T + Z engineering ζ Benny and -2804,6265[6. The above cells were labeled with Indo-1, 5μT3μM and 80HDPAT. In the presence or absence of 10nM, in the presence or absence of 1100n subiveron. The intracellular Ca concentration was monitored for 5 minutes. Intracellular results The concentration value of Ca++ and (2) are shown. On the other hand, c A M P is It was extracted from cells and the value was quantitative as described. In this case, Adeni All cells were treated with 10 μM forskolin to activate lactate cyclase. (forskolin). PMO 1 (cAMP/106 pieces) cells).

Production of 5HT and 5HT1a specific agonist 80HDFAT in T cells Comparison of physical and pharmacological responses Biological responses Pharmacological responses EC50IC50IC50 Ligand ca++ cAMP Ca++ cAMP 1E button 9)-80HD PA, T 5 days M 3 days M 1.5 days M5 HT 200nM 400nM 3 ．． OnM Subiveron 60nM 70nM 50. OnM cell intracellular Ca+ level determination was carried out using Jurkat cells, and c A and M P were measured using activated T cells. I went there. Furthermore, the Jurkat membrane was treated with 80 HD P AT of M for one day. Labeling and displacement treatments were performed in the presence of various concentrations of the given ligand.

5μ plate (7) plate in 2- quiescent PBL in the presence or absence of PHA, PWM or 0KT3; Cultured at various times, harvested and analyzed for 5HT content. These results are shown in Figure 8. Shown below.

PBL contained 5HT at 4 μmol/10 cells at the early stage of culture. concerned Levels were maintained for 4 days or disappeared by day 7. On the other hand, 0KT3 or PHA The 5HT content decreases to 75% or more within 72 hours due to stimulation of PBL by either of the following: 5μT receptors had reached maximum levels at approximately that time. Also, P When PBL is stimulated by WM, the level of 5HT is approximately 50% for the first 48 hours. It increased until then, and then gradually decreased. In addition, these cells are approximately 7 days old. It was maintained in 2pmol5HT/10 cells.

Once the PBL is increased, the increase in PBL stimulated by the Growth is partially inhibited by relatively high concentrations of 5HT. Figure 9 shows this finding. and also the P B against other T cell mitogens 0KT3 and P W M The effect of 5HT on the proliferation of L. That is, in the presence of 0KT3 The proliferation of these PBLs is not affected by the addition of 5HT. On the contrary, PW Addition of 5HT to M-stimulated cultures increased H-TdR uptake by 3-fold. Ta. Moreover, half of the maximum uptake increase corresponded to 5 μT and 15 μM. Also, This phenomenon can be observed when 5HT is added from day 3 to day 5 of the culture period. At this point, 5μT receptors are maximally expressed (see Table V). In addition, the culture period Addition of 5HT early in the period increases proliferation three-fold; More 5HT (100 μM) is required for this purpose. To explain this difference One possibility is that the 5HT activity is due to the 5μT receptor, which does not appear until the third day. mediated by the metabolism or oxidation of 5HT at the beginning of the culture period on day 3. reducing the effective concentration of 5HT in the culture medium by Conceivable.

Furthermore, it binds to the 5HT receptor and interacts with the 5μT receptor on activated T cells. Proliferation of PBL stimulated by PWM as described above for interacting analogues of were tested for their ability to promote or inhibit Of these tested, 5HT, Ketanserin (5HT2 receptor antagonist) and alpha methyl serotonin (5HT2 receptor agonist) inhibits the proliferation of T cells at a concentration of 10 to 30 μM. The range was increased approximately three times. Shigashi and two other 51 (T2 receptor antagonists) agonists, namely ritanserin (selective for 5HT2) and mian Serine (selective for 5HT2 and 5HT1c) stimulates the T cell proliferation. There wasn't. However, ritanserin showed some inhibitory properties at high concentrations. It seemed like a sea urchin.

These results are shown in number 10. Next, CD4+ and CD4+ in culture of PBL Proliferation of 8+ T cells was stimulated with PWM. In this case, CD4+ and CDS+92 In the mixed culture of 8 cells, the cells of CDS+928 cells mainly showed higher growth on the 7th day. It was being proliferated. The results shown in Figure 5 also indicate that if the CDS+928 spheres PBL culture with 5HT or 5μT receptor ligands when removed by complement treatment. This indicates that the ability to stimulate the growth of nutrients is lost.

The above results also indicate that T cell embryos express 5μT receptors and that either 5HT or PWM Enhances stimulated T cell proliferation and shows that the T cells contain 5HT. are doing. This means that the 5HT is normal in T cells activated by PWM. This indicates that it may be important for proliferation. In this case, tryptophan Inhibitors of hydroxylase, which inhibit the rate-limiting step in 5HT synthesis in cells It was used to deplete 5HT accumulation.

The results in Table 9 are for 5HT synthesis with p-chlorophenylalanine (pCPA). show that inhibition of PWM also partially inhibits cell proliferation stimulated by PWM. . Furthermore, when 5F(T or ketanserin is added to these cultures, the pCPA The inhibitory effect of is reversed.

Table 9 Suppression of T cell proliferation by inhibiting 5HT synthesis PBL PWM pCPA 5HT Ketanserin 3H-TdR--1---℃μ head μ+massage bar 10mm) military bar －－＋　－－－－1728±210 ＋　＋　---11954±1022 10 + + −-4,306± 688+ + + + 9726±1614 ±+++10068±469 CD8 + 1° Resosa T plate heated by pWM, 5HT, - CDS+ suppressor T cells are the most proliferating cells in PWM-stimulated PBL cultures. It accounts for the largest proportion. Inhibition of 5HT synthesis by pCPA was stimulated by PWM. In order to inhibit proliferation in culture, the pCPA inhibits expression of suppressor cell function. It is also thought to inhibit These results are shown in FIG. Reduced 5HT content To do this, PBL cultures were incubated with PW in the presence or absence of pCPA. Stimulated by M. In addition, agonists for 5HT receptors can be produced to To reverse the effects of PA, some cultures were treated with ketanserin. difference Furthermore, after 7 days, cultures were washed, treated with mydomycin C, and stimulated with PWM. Suppressor cell activity was tested by adding fresh CD4+ T cells. As a result, in PWM Cells corresponding to the stimulated cultures showed proliferation of CD4+ T cells in the presence of the PWM. Suppression was achieved up to 70% by setting the ratio of suppressor cells and corresponding cells to 1=2. . In addition, inhibition of proliferation by up to 50% was achieved with 7 x 10 cells or 1 suppressor. This was achieved with 7 corresponding cells per cell. Furthermore, the suppressor details The presence of pCPA in the main culture used to generate cells indicates that the suppressor Cell activity was completely inhibited. Meanwhile, ketanserin was cultured with pCPA. When added to the body, the activity of the suppressor cells was normal in terms of regulation.

Other T cell mitogens, such as PHA or 0K73, target CD4+ and CD8+ T cells. stimulates both proliferation of cells, but within the first week of culture, CD8+ T cells However, it is not possible to express the activity of suppressor cells. Furthermore, said 0KT3 or P PBL stimulated by any of the HAs loses its 5HT content within 72 hours. PWM-stimulated cultures retain the 5HT for at least 7 days, although the amount decreases. Maintain content. Also, stimulated by either PHA or 0KT3 Addition of pCPA to cultured cultures did not inhibit growth (not shown). In addition, applicable To investigate the relationship of 5HT content to suppressor cell activity, PBL was Ketanserin, a cellular 5HT receptor agonist g) Stimulate with 0KT3 in the presence or absence of suppressor after 5 days of culture. Cell activity was tested. As a result, T cells obtained from the culture stimulated with the 0KT3 There was no detectable suppressor cell activity in the embryos. On the contrary , T cell embryos obtained from cultures stimulated with both 0KT3 and ketanserin , expressed suppressor cell activity when tested in secondary culture. These consequences The results are shown in Figure 12.

Mitan J-±T lymphocytes EU responds to 5HT as a mitogen Proliferation of CD8+ T lymphocytes in response to PWM requires CD4+ T cells. the above A 3-fold increase in PBL growth cultures with 5HT or 5HT receptor ligands It disappears after removal of 8+ T cells. Therefore, 5HT acts on the CD8+ T cells. Acts as a co-mitogen and in the presence of PWM It is thought that it has the potential to stimulate proliferation. These results are shown in FIG. Na The culture of CD8+ T cells was cultured in the presence of PWM or in the presence of PWM and IL2. It does not grow below.

In contrast, CD8+ T cell-containing cultures and PwNl Proliferates in the presence of encerine. Furthermore, the degree of proliferation is such that CD4+ T cells 8+ when added to a culture of T cells. Also, 5HT or Ketanse Addition of phosphorus alone does not stimulate proliferation of the CD8+ T cells. Above all, this data show that 5HT is a common mitogen for CD8+ suppressor T cells. This is considered to indicate that

Activation by 5HT cAMP levels in specific 5HT receptor subclasses Two signal transduction pathways linked to the adenylate cyclase denaturation and activation of phospholipase C. In this case, the phosphor Activation of ZeC increases inositol phosphate and increases intracellular Ca+ concentration. Ru. In addition, in Jurkat cells, 5HT interacts with the inositol phosphate. Promotes an increase in internal Ca+1 concentration. Therefore, the level of inositol phosphate and Changes in intravesicular Ca + + were measured in T cell embryos (PHA) in response to 5HT, and , the change was measured as a positive regulation in response to 0KT3. These results are shown in Table 7. Shown below. Compared to Jurca Soto cells, 5HT showed a change in the amount of inositol phosphate mentioned above. and does not cause changes in intracellular Ca+ concentration. Also, as a positive regulation, 0KT 3 promotes increases in the concentration of inositol phosphate and intracellular Ca+ concentration in T cell embryos do. Therefore, the T-cell tumor is affected by the level of inositol phosphate and intracellular Ca. Since the amount of intracellular cAMP does not respond to 5HT due to a change in concentration, the amount of intracellular cAMP Comparisons were made in the presence and absence. i.e., resting T cells, T cells Comparisons were made between three types of tumor and Nyurkat cells, and the CAMP level was determined by folding. A comparison was made in the presence and absence of skolin. These results are shown in Table 8. .

As a result, resting T cells do not raise intracellular cAMP levels in response to 5HT; , activated T cells more than doubled in response to 5111° (5 μM). increased. In addition, Nyurukanoto cells change cAMP levels in response to 5HT. (, there was no activation of forskolin or adenylate nucrase, It is thought that intracellular cAMP was increased. Therefore, forskolin is cAMP concentration was increased in cells, activated 'r cells and Jurkat cells.

The forskolin-dependent increase in cAMP amount is due to the addition of 5HT to activated T cells. However, addition to resting T cells or Jurkat cells inhibited the inhibition. No adverse reactions occurred. Figure 2 shows the increase in cAMP levels in activated T cells. It shows the concentration of 5HT required to cause Figure 2 shows a decrease in cAMP levels in the activated T cells. The cAMP The change in level is proportional to 5HT concentration ranging from 30 nM to 3 μM. Ma In addition, the value of half the maximum response corresponds to a 5HT amount between 200 and 800 nM. , this is similar to the case of the Kd value obtained from the combined data. Also, PM Addition of A also increased cAMP levels in the T cell tumors and Jurkat cells. Ras. However, compared to forskolin, 5HT is effective against all cell types. However, the PMA-dependent increase in cAMP is not inhibited. These results are also shown in Table 9 above. Changes in intracellular cAMP concentration are known to affect activated T cell function and proliferation. (53) Based on the above results, 5HT has different effects on activated T cells. In subpopulations or activated T cells affected by different stimulatory factors, It is believed that different levels of intracellular cAMP can be regulated. The results shown in Table VII are P cAM in T cell tumors initially stimulated with HA, OK, T3 or PWM A comparison of changes in P concentration is shown. The T cells were harvested at the peak of the proliferative response and cAMP content was assayed in the presence or absence of HT. In addition, the culture Cultures were cultured in the presence of 5HT for the last 48 hours to determine the effect on proliferation. . As a result, 5HT doubled the level of cAMP in T cells stimulated with PHA. and slightly increased the amount of cAMP in cell cultures stimulated with 0KT3. However, in T cells treated with PWM, the amount of cAMP was reduced by 60%. . In other words, T cell culture with different stimulatory factors in the presence or absence of 5HT It was found that the proliferative response in cells affects the level of intracellular cAMP. Therefore Therefore, 5HT slightly inhibits T cell proliferation in response to PHA or inhibits T cell proliferation in response to PWM. Promotes the proliferation of T cells by more than 3 times. In addition, prior to stimulation by the PWM Depletion of CD8+ T cells by The effect as a togen disappears. Therefore, 5. HT is c A M P level Does the regulation clearly promote or suppress T cell proliferation? can. However, the direction of the response depends on the mitogenic action and the above stimulating factors. It is thought that this is determined by the subpopulation of T cells that respond to this problem. Figure 3 is The results show that the proliferation response of PWM-activated T cells to 5HT is relatively accelerated. Ru. Furthermore, the promotion of proliferation in the presence of cough 5 HT was detected within a few hours, and 40 reach its maximum peak in time. In addition, the level of NIP to C was determined by the control amount at 30 minutes. 60% of the control amount, and in the subsequent 40 hours, the control amount decreased by 40%. Ru. Figure 4 shows the effect of 5HT as a common mitogen on the PWM activated T cells. It shows the concentration dependence of

In this case, 5HT was added to the cultures 48 hours before the proliferation assay. In addition, the maximum Growth promotion was observed at 30 μM of 5HT, and the IC50 was 10 μM.

The outcome of adding 5HT to T cells depends on the phenotype of the responding cells. Sunawa Furthermore, promotion of T cell proliferation by 0KT3 or PHA increases CD4 and CD8 levels. A current T-cell tumor is generated, and promotion of T cell culture by PWM results in a CD8-phenotype T cell tumor. Cytomas primarily occur. Note that the responses of these two cultures to 5HT are different. . Additionally, addition of 5HT to cultures stimulated with the PHA or 0KT3 increases the amount of cAMPO and slightly inhibits the T cell proliferation. On the contrary , addition of 5HT to PWM-stimulated cultures decreased cAMP levels by 60% and T Increases cell proliferation 3-4 times. In addition, cell proliferation in these cultures is based on FM. As measured by S (not shown), CD8+ T cells were predominant. , express suppressor cells but do not exhibit cytotoxic effects. Also, CD8+Tl 1B cells (thereby acting as a common mitogen for 5HT in these cultures) effect disappears. The 5HT can also be used to target certain types of cells such as smooth muscle cells or fibroblasts. Acts as a growth factor for cells (46.47). This indicates that the growth of the 5HT This is thought to indicate that its properties as a factor are not limited to immune system cells.

From the above, T cells express receptors corresponding to 5HT when activated, and 5HT is shown to affect activated T cell proliferation and function. Furthermore, 5HT is the main component of platelet secretory granules and is therefore likely to be released at sites of inflammation. It has been revealed that (2-4).

Therefore, the 5HT is present at sites of inflammation and influences T cell function. it is conceivable that. Furthermore, it has recently been shown that purified resting T cells also contain 5HT. has been revealed (50). Then, the T cells respond to gamma FN and become mediators. Releases 5HT throughout the body. Note that under such conditions, the concentration in the medium is approximately 3oonM/ml/10 T cells. This means that T cell 5H This is very similar to the case of the Kd of the T receptor. And by 5 HT or T cells Once released, the 5HT plays an important role in the regulation of T cell function by T cells. They begin to fulfill their duties. Furthermore, CD8+ T cells are activated in the absence of CD4+ T cells. It has been shown that P.W.M. does not proliferate in response to PWM (54-56). . Therefore, it can be released by CD4+ T cells in response to 5HT or gamma IFN. , and the hypothesis that it acts as a growth factor for CD8+ T cells becomes interesting.

Rheumatoid arthritis is a major disease that forms a large group of rheumatic symptoms.

Rheumatic symptoms with autoimmune components include rheumatoid arthritis and systemic erythema. Sexual lupus, Sjogren's syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter's syndrome, and Besse's syndrome. An example is Behcet's disease.

The arthritis in rheumatoid arthritis is caused by destruction of the joints accompanied by postfactorial deformities. arise. In addition, the affected areas are not limited to the joints, but also vasculitis caused by immune syndrome. This includes the skin, eyes, and lungs.

This arthritis is caused by synovial fluid cells and various cells that penetrate into the synovial layer of the joint from circulating body fluids. caused by complex interactions with the elements (and their soluble products).

This causes massive cell proliferation and also leads to activation of the synovial fluid cells. . Furthermore, the properties of the synovial fluid cells in tissue culture are similar to those of transformed cells. has been equated with. In addition, a detailed discussion of the series of effects that lead to joint disorders is provided. The theory is Fundamental Immunology 2ed, P. aul, (1989) pp 846-849).

Subjects with experimental arthritis include adjuvant, a purely T cell-mediated autoimmune disease. Bunt's arthritis was selected (also note that this arthritis is caused by mycobacterium condensation in oily fluids). Rats by injection of Mycobacterium mycobacterium (complete Freund's adjuvant) It can be induced by muscle tension in (eg Lewis rats). Na Mycobacterium peptidoglycan and proteoglycan in joints There are structural similarities between the two. In other words, the Mycobacterium tuberculosis The peptide contains an epitope recognized by T cell-mediated adjuvant arthritis. be caught. In addition, T cells from a parent with rheumatoid arthritis share this common epitope. Reply to Tope. See above (Paul).

Harm J: Unjuvant B: Heat-killed Mycobacterium butirirum (Myc.

bacterium butyrium) or Mycobacterium tuberculosis H Complete Freund's adjuvant by adding extra heavy mineral oil with 37Ra, 10mg/ml (CFA) was created. On day 0, female Lewis rats (200-225 g) 0.1 ml of the adjuvant was subcutaneously injected into the sole (administration site) of the right hind leg ( 100 μg/animal). First, an initial inflammatory response occurred in the treated paw.

This symptom subsides by the 5th day, with secondary ongoing inflammation/inflammation occurring from the 9th to the 12th day. Symptoms of inflammation occurred in both the treated leg and the untreated left leg. Behind the pacification and heel diameter measurements for initial symptoms on the 2nd day and secondary symptoms on the 12th and 16th day. (both feet) using hand-held engineer's calipers. Set the measurement result to 0 Change in swelling relative to the diameter of the foot on day + or - expressed as the mean value of S, E, did.

The drug is then mixed with water, saline, or either of these with 2% polyethylene glycol. Cole 400 10.1% Dissolved in a mixture with Tween 80 or Suspended and administered by regular means (orally, intravenously, intramuscularly) before or after the onset of symptoms. gave.

As a result, the adjuvant arthritis is clearly mediated by T cells and the disease state We were able to obtain activated T cells that can be transferred to healthy animals. In addition, Tanserine inhibits the progression of the disease at 10 or 30 mg/kg, It was found that ketanserin did not prevent the progression of the disease state.

Table 10 Adjuvant R; 1 Tanserine Data Experiment 1 2 Symptoms - Day 16 Average heel diameter % inhibition state ±S, Emm Control 2.55±, 44 contrast extremely + Ritanserin 0.93±, 3163.5 Activity 30mg/kg i, p.

(Number of days - 1, 0, 1, 5, 7, 9, 12) Ten ketanserin 2.09±, 462 3.7 ＼1 Experiment 2 Control 2.35±, 56 contrast + Ritanserin 1.39±, 3040.3* Activity 30mg/gp.

(Number of days - 1, 0, 3, 6, 8, 10, 13) + Perancerin 2.44±, 34 4.3 ＼Activity experiment 3 Control 3.28±, 44 contrast +ritanserin 2.33±, 4329.0 inactive 3.0mg/kg +), o.

+ Ritanserin 0.89±, 2172.9 extremely 10m k, o, ’- *The difference in activity can be explained by a slightly different dosing schedule.

Purified recombinant human IFN was obtained from Amgen (Thousand). 0aksSCA) and diluted with vehicle before use. 5-0H-) Rib tophan, 5HT,) ribtophan, 5-0H-indoleacetic acid and melatoni p-chlorophenylalanine (pC) was obtained from Sigma (Si gma). Research Biochemical Co., Ltd. (PA) ls, Inc.) (Natick, MA). Also, RPM1164 0 medium, bovine fetal formation (Fe2), sodium pyruvate and sodium glutamate. Thorium was obtained from GIBCO. flat bottom multiwell tissue culture dish Becton Dickinson or Costa (Costar). Furthermore, the solvent for HPLC is Aldrich (A Obtained from l　d　r　ic　h).

1 red light 1 The human cervical carcinoma cell line ME180 was collected from the American Type Culture Collection ( Amer　　　　can　TypeCulture　Co11ection)( Rockville, MD) and in tissue culture flasks. RP in the presence of 0% FSC, without antibodies, under human body conditions at 37°C in air containing 5% CO2. Maintained in M11640 medium.

Furyanobu Eiji A ME180 cells were grown in complete medium at 1X105 cells/ml.100 μm/well ( 96-well plate) for 3 days in the presence or absence of different amounts of IFH. It was cultured for a period of time. On day 3, the culture was treated with 1 μCi of “H-TdR” for 5 hours and Collected on filter paper. The culture was performed in duplicate at least three times and similar results were obtained. . Most were also repeated as positive controls for the next experiment. Create double were equal within 10% of each other. In addition, 3■ (-TdR uptake is Detected by body scintillation spectrometer.

HT of and Cells and media after various treatments at various times as described in the text. It was separated and mixed with ethanol to give a final concentration of 70%. Then set the sample to 0 After overnight storage at −4°C, the precipitate and cell debris were removed by centrifugation. So Afterwards, the sample was concentrated using a Speed-vac. It was dried, further dissolved in 20 μm buffer yA, and analyzed by HPLC (Hewlett-P Ackard) (13).

The HPLC device is equipped with a 280nm excitation filter and a 360nm emission filter. Equipped with a fluorescence detector (Gilson). In addition, the two types of mobile phases are A diphosphoric acid 50 nM of triethylamine adjusted to pH 3.0 and B: containing 40% methanol. It consists of a buffer A and a buffer A. Furthermore, the elution gradient is from A100% to B100%. Linear over 25 minutes. Known standard retention times are 3°50 minutes at 5HT, 5 -0H-4.77 minutes for tryptophan, 9.11 minutes for tryptophan, 5-0H - Indoleacetic acid took 12.51 minutes, and melatonin took 21.69 minutes. . In addition, the identification of peaks in cell extracts using known standards can be performed using known amounts of standard substances. by mixing with cell extracts and demonstrating the identification of peaks on the chromatogram. So I went. The recovery rate of tryptophan and 5HT from the cell extract was determined by the extraction process. A value determined by mixing the cells with a known amount of standard substance before starting the treatment. It was over 90% of the time. The quantitative treatment of 5HT and tryptophan was carried out three times. We obtained similar results.

IF by tryptophan or 5H tryptophan" Inhibition of cell proliferation by IFH is associated with indoleamine 2 in certain tumor cell lines. ．． This is due to loss of tryptophan promoted by 3-dioxygenase. Ru.

Therefore, addition of exogenous tryptophan reduces the inhibition of cell proliferation by the IFN. (Reference 6-9 and Figure 1). The tryptophan is an essential amino acid, Required for protein synthesis and also for the synthesis of neurotransmitters 5HT and melatonin It is. The results shown in Figure 1 indicate that the tryptophan and 5I (tryptophan or has been shown to reduce IFN-mediated inhibition of cell proliferation. In terms of molecular structure, the 5H) Ribtophan is thicker than tryptophan (the effect of IFN on ME180) It is 500 times more effective than tryptophan in inhibiting the activity (17B figure). In addition, the 5H tryptophan is a precursor in 5HT synthesis, and is a precursor in protein synthesis. It is not used for construction. This indicates that the inhibition of 5HT synthesis was caused by IFN-treated cells. We show that this may be an important consequence of tryptophan depletion in cell culture. ing. Results in Figure 2 are for comparison of IFN-mediated inhibition of cell proliferation. . Optimal results are obtained when the compound is added on days 1 and 2 of the 3-day culture. obtained if Note that under these conditions, only 5H1-ributophane inhibits IFN activity. In addition, other 5) (tryptophan metabolites, 5HT .

Melatonin, N-Ac-5HT or 5-0H-indoleacetic acid is 5H trypto It induces FAN metabolism but does not inhibit the IFN-mediated inhibition of cell proliferation.

From the above results, the loss of tryptophan in the culture medium is caused by 5H tryptophan or or its metabolite 5H'r, resulting in inhibition of cell proliferation. vinegar. Figure 3 shows intracellular tripod levels at different times in culture in the presence of IFN. Levels of tophane and 5HT are shown. That is, the tryptophan and For cells containing both 5HT and 5HT, both of these can be cultured in the presence of IFN. and lose money. Furthermore, 5H tryptophan could not be detected in the cell extract. It was. In other words, the synthesis of 5H tryptophan is the rate-limiting step in 5HT synthesis. Therefore, the 5H)ributophane is generally not accumulated within cells.

5H) 5HT concentration of IFN and proliferation cycle due to liptophan To see if 5H tryptophan restores 5HT levels, tumor cells were In the presence of an inhibitory amount of IFN, 5 μg (incubated in the presence or absence of tryptophan) (Table 5). Treatment with IFN reduces 5HT to undetectable levels; When H-tryptophan is present, the level of 5HT is maintained. actual , in cultures containing the IFN and 5H tryptophan, compared to untreated cultures. , 5HT levels were four times higher. Note that even if 5H) liptophan is added, this We were able to restore cell proliferation in these cultures.

Man H tryptophan exists in IFN "Sip" Kame 1 Ritsu φ2-Kari 9"- Length Gutphan 　L Shuko Regarding the ability of 5H1-lyptophan to block IFN-mediated inhibition of cell proliferation. There are two likely explanations, one is that it is a loss of tryptophan from the culture medium. Another reason is that it is intended to prevent intracellular 5HT levels. This is to recover Bell. The results in Figure 4 are processed using IFN. 5H tryptophan prevents the loss of extracellular tryptophan in cultured cells. It shows that it does not. For control cultures of ME180 cells, 72 hours of culture In nutrition, no significant amount of tryptophan is consumed. On the other hand, treated with IFN In cell culture, 50% of tryptophan is consumed in 24 hours; 95% is consumed within hours. Furthermore, the culture medium in the culture treated with said IFN The rate of loss of tryptophan in is reduced by the addition of 5H1-lyptophan I couldn't. However, under these conditions, the levels of 5HT and Cell proliferation returned to control values.

J’fil multiplication by f4 tryptophan! 5 H to boot fan reduced by Growth of tumor cells in the presence of various concentrations of methionine or leucine was shown in 1. Maximum rate is reached at 0-20 HM amino acids. On the other hand, various concentrations Growth of tumor cells in the presence of tryptophan at 50-100 μM maximum velocity is reached in noic acids (6). Figure 5 shows different concentrations of tryptophan M performed in the presence or absence of 3 μM 5Hl-ributophane. A comparison of proliferation of E180 cells is shown. Also, in the absence of 5H tryptophan In this case, maximum proliferation occurred in the presence of 50 HM tryptophan. Also, 5H In the presence of tryptophan, maximum proliferation occurs in the presence of 10 HM of tryptophan. Ta. From these conditions, the addition of 5H)ributophane increases the cellular 3H-tryptophan content. It was found that this did not affect the ability of the protein to be incorporated into proteins. This means that the 5H tri- Ptophan is not converted to tryptophan by ME180 cells, and protein It also indicates that it is not used for synthesis (not shown).

5HT order plate M E 180 Proliferation by fraud and - The above results indicate that a decrease in intracellular 5HT inhibits the proliferation of ME180 cells. It shows. Therefore, ME1870 cells were treated with tryptophan hydroxyl. 5HT in the presence of pCPΔ, a specific inhibitor of 5HT. We investigated whether this resulted in loss of cell growth and inhibition of cell proliferation. The results are shown in Table 6. pCP Culturing ME180 cells for 48 hours in the presence of A also resulted in loss of tHT and inhibition of cell proliferation. However, no loss of extracellular 1-ributophane occurred. Furthermore, the said 5 Loss of HT and inhibition of cell proliferation was prevented by addition of 5H tryptophan .

Therefore, two independent mechanisms, namely indoleamine-2,3 - Tryptophan oxidation catalyzed by dioxygenase or tryptophan Depletion of 5HT by inhibition of hydroxylase also inhibits tumor cell proliferation. I got it. Also, in both cases, the growth of the tumor cells is due to 5H) liptophan Recovery occurred due to recovery of 5HT levels.

Other additional tumor cell lines, as well as normal diploid cells, may also be affected by 5HT. Contains or pcPA reduces intracellular 5HT concentration and inhibits cell proliferation. Furthermore, 5H) Libutofun restores 5HT levels and stimulates the cell proliferation. was tested to see if it could reverse the inhibition of These results are shown in Table 7. this The results showed that all tumor cell lines tested contained 5HT, and four of the five cell lines proliferation was inhibited by cCPA. Furthermore, the growth inhibition of the tumor cell line was 5 The addition of H-tryptophan significantly reversed the effect. This is true for many tumor cell lines. , that 5HT or a metabolite of 5HT is required for its proliferation. It shows. In contrast, several different types of normal diploids have cCPA Therefore, it was not hindered.

Table 5 5H tryptophan (5Htp) is 5H tryptophan (5Htp) in IFN-treated ME180 cells. Restores T levels and their proliferation.

Intracellular level* Culture conditions 5HT growth Mouth V "Ga1-roe--"-LiL Niko-1" (c)-ME180 cells 18 98 069±1025+IFN, 1 19789±874+IFN+5Htp 80 99672±1984Ht32 96331±l593 *ME180 cell culture was carried out for 3 days and inhibited to 2X105 cells/ml. I FN (300u/ml) was added initially, and 5Htp (final concentration = 3μM) was added every Added daily.

Then, a sample was taken and analyzed by HPLC for 5HT tryptophan hydroxyl. Inhibition of silase reduces 5HT levels and inhibits cell proliferation.

Culture conditions Intracellular water Extracellular* Growth 5HT Tryptophan (3H-TdR) (molm (m ME180 cells 16.5 12 44443±2244+p CP A 3 ．． 1 25 16642±1723+pCPA +5Htp 37.1 Undetectable 42221±4128+ 5 HtJ2□- -Yu ↓-1 trace\　42982±2629*NiE180wI cells to pCPA2m Cultured for 48 hours in the presence of 10 μM M and/or 5H tryptophan. culture Cells or cells were collected and analyzed for tryptophan and 5HT by HPLC. Ta.

Inhibition of 5HT synthesis inhibits proliferation by tumor cells.

Cell 5HT content proliferation (cpmXlooo) population (mol/mg protein) Control +pCPA +pCPA+5-0H-tyrp Bile] KoL exchange JJL type E180 13±2 44,7 12,6 42.2T29 24±3 56,7 14.1 47.4 31±3 85.6 24.0 40.2M OL T-4 22±5 34.3 36.5 31.539±6 194.0 71.8 1 55.6T20 Undetectable 29.0 6.5 29.7 3 times ≧ 1 cell T cell embryo 25±2 45.9 37.1 41.4 fibroblasts 29±4. 10.3 9.3 10.2 (lungs) fibroblast Undetectable 4.8 3.9 4.5 (synovial fluid) endothelial cells \　7.5　7.9　7.5 *Different human cell lines were obtained from ATCC. These culture conditions are the same as those already described. (6) Serum was dialyzed before use to remove 5HT. ME180 (cervical carcinoma cells), BT20 (thoracic carcinoma cells), HT29 (colon adenocarcinoma cells), U937 (histological lymphoma, monocytosis), Julcasoto (T-cell lymphoma cells) , MOLT-4 (acute lymphoblastic leukemia), endothelial cells (subculture), synovial fibers Blasts (subculture), lung fibroblasts (MRC-5; long-term lineage) and T cell tumors The primary culture was cultured for 48 hours.

Serotonin has been shown to be present in malignant or tumor cells, and 5HT Two similar receptors were found to be present on tumor cells. Furthermore, the tumor cells increase. Activated T cells as well as serotonin receptor agonists and antagonists It has been shown that it can be regulated by serotonin synthesis or by inhibition of serotonin synthesis.

In nervous tissue, serotonin (5-hydroxytryptamine, 5HT) is Synthesized by hydroxylation and decarboxylation of liptophan and accumulates in granule tissue (35-36). Furthermore, the 5HT is released by appropriate stimulation and is binds to specific receptors on cells and activates the secondary messenger pathway (37-39 ). The rate-limiting step in serotonin synthesis is the activity of tryptophan hydroxylase. level. Furthermore, a specific enzyme inhibitor of the tryptophan hydroxylase Bitter, or p-chlorophenylalanine (pCPA), is a cellulose It has been used to deplete levels of tonin (40-41). Therefore, humans Cervical carcinoma cells (ME180) in the presence or absence of 5-0H-)ributophane The cells were then treated with pCPA for 48 hours, and the 5I (T content and rate of cell proliferation were examined).

As a result, in the cultured cells, 13±2 pmol/mg protein of 5HT was transferred to pCP. After treatment with A, it decreased to 4±1 p m o I/m g protein. Similarly, the applicable In the culture medium of cultured cells, 44 nM of 5HT is reduced to 10 by the action of pCPA. decreased to nM. However, addition of 5-0H-)-ributophane caused control cells to I was able to recover 5 HT to the level of 5 HT. In addition, pC of the ME180 cells Treatment with PA reduced H-TdR uptake by 70%. Furthermore, the said Growth inhibition was also significantly reversed by 5-0H-tryptophan (Table VI ). Also, the 5HT content of some other tumor cell lines and normal diploid cell lines were also investigated and cultured in the presence of pCPA for 48 hours to reduce 5HT levels. Tried a little (Table IX). As a result, the concentration of 5HT in the extract of the cells was The concentration ranged from 10-40 pmo I/mg protein. In addition, the precursor of 5HT 5-0H-1-lyptophan could not be detected in the cell extract. difference Furthermore, in cultured cells in the presence of pCPA, 5 of the 6 tumor cell lines tested Cell proliferation was inhibited in normal diploid cells, or there was no significant effect in normal diploid cells. was not seen. Additionally, 5-0H-1-reactive cells were added to pCPA-treated cell cultures. Addition of putophane significantly reversed growth inhibition in tumor cell cultures; , no particular effect on cell proliferation was observed in normal diploid cell cultures.

Therefore, depletion of 5HT leads to inhibition of tumor cell growth, but normal cell growth There was no such effect, and furthermore, the addition of 5-OH-1-ributophane Restoring 5HT levels restores proliferation in these cells.

Serotonin accumulates in the culture medium to various concentrations and is released through 5HT receptors into secondary stimulants. (37-38). In addition, the secondary Metsenger 5 that transforms the tract (release of inonthole phosphate and changes in intracellular Ca+) HT receptors are identified on gel-cut cells, but MoI t-4 cells - Not done in L (42). Therefore, many 5HT receptor agonists and antagonists are available. Test antagonists to see if they inhibit the growth of the tumor cells as opposed to 5HT. I looked into it. Among those tested, the 5HT2 receptor antagonist 40 Nserin inhibited tumor cell proliferation (in the six lines tested) in the range of 10-50 μM. (in 5 species) 70-95% transcription or normal diploid cell growth did not interfere. However, at low concentrations (<10 μM) of ritanserin, tumor cells growth was not inhibited. In addition, when 5HT is added, the ritanserin The inhibitory effect was partially reversed (Table X). Furthermore, Mo1t, a type of tumor cell, -4 proliferation was not inhibited by pCPA and was also inhibited by ritanserin. It wasn't done. In addition, pCPA reduces the concentration of 5HT in cell extracts, but Ritanserin substantially increased the 5HT content in the cell extracts. for example , the extract from ME180 cells contains 5HT of 15.5 pmo and 17 mg protein. However, the extract from the culture treated with ritanserin contained 58.8 pmol/mg protein. Contains white 5HT. Therefore, inhibitors of 5HT synthesis and 5HT receptor antagonists Both antagonists inhibit proliferation by tumor cells, but inhibit growth by normal diploid cells. Does not inhibit growth. Furthermore, various types of ligands that bind the 5HT receptor were tested. and investigated their ability to prevent ritanserin-mediated inhibition of tumor cell growth.

These results are shown in FIG. According to this, another 5HT2 receptor antagonist ketanserin and perancerin, 5HT1c receptor antagonists mianserin, and 5HT1 receptor agonists and antagonists 8-0H-DPAT and bropranolol are each treated with ritanserin. The inhibition of proliferation was partially prevented in a concentration-dependent manner. These data are Roughly equivalent to the data for Jurkat cells, but with these 5HT receptors It was not possible to classify the 5HT receptor subtypes characterized by type (42 ). It is interesting to note that ritanserin, ketanserin, and peranserin all 5HT2 receptor antagonists with similar pharmacological and biological properties On the other hand, ritanserin inhibits tumor cell proliferation, and ketanserin and nserin is to block the inhibition by ritanserin. This means these This means that the data are sufficient to define a new 5HT receptor subtype. It's not about doing it. In order to completely define the types of these 5HT receptors, it is necessary to More detailed analysis is required for these receptors. Also, these data The 5HT and 5HT receptors are associated with certain types of tumor cells in tissue culture. It is thought that the possibility that this is necessary for the propagation of the system has emerged. The characteristics of this type of growth include , a growth factor for the cell that binds to the cell surface receptor and stimulates cell function. The autocrine pathway of growth factor action, which releases There are some consistent results (43-44). In the data shown here, the 5HT has been shown to be produced by tumor cells and required for proliferation. moreover, Studies of ligands known to bind to the 5HT receptor have shown that 5HT is It has come to be thought that it has the effect of stimulating proliferation through cell surface receptors.

From this point of view, it can be seen that the 5HT is responsible for both inactive fibroblasts and smooth muscle cells. It is important to know that this has been shown to be a long factor (45-47).

In these instances, exogenous 5HT versus endogenous 5HT promotes DNA synthesis. used to. Furthermore, these results demonstrate that normal diploid cells can be transformed by acquiring the ability to produce growth factors or growth factor receptors. This is consistent with many current concepts of tumorigenesis (48-49). vinegar That is, the production of 5HT and 5HT receptors is a permanent transformation of normal cells. This can be one way to obtain the current model. The tumor cells then release serotonin. It remains unclear whether the serotonin is synthesized and required for proliferation in vivo. This is an important problem to solve.

Culture 5HT growth Intracellular Extracellular (cpm) −〇■Dango noodles (nM) ME180 cells 13±2 44±3 44700±5620+pCPA 4±1 10±2 12600+1220+pCPA+5-0H-tryp 16±4 56±4 42200±4220 Table 12 5 Inhibition of HT synthesis inhibits proliferation by tumor cells but not by normal diploid cells Does not inhibit proliferation. Different types of human cell lines were obtained from ATCC. these The culture conditions are as shown in (50). First, before using to remove 5HT Serum was dialyzed. ME180 (cervical carcinoma cells), BT20 (thoracic carcinoma cells), HT29 (colon adenocarcinoma cells), U937 (histological lymphoma, monocytic type), Julka (T-cell lymphoma cells), MOLT-4 (acute lymphoblastic leukemia), endothelial cells (-subculture), synovial fluid fibroblasts (-subculture), lung fibroblasts (MRC- 5: long-term series) The primary culture of T cell tumor was also cultured for 48 hours. Other conditions are in Table 1 As described in 1.

5HT content proliferation Cells (pmol/mg protein) (cpm x 1000) Population Control +pCPA +cCPA + p CP A + 5-OH- r Bile JLj = Twisted JJL series E180 13±2 44.7 12.6 42,2T29 24±3 56.7 14.1 47.4 31±3 85° 6 24,0 40,20LT-4 22±5 34.3 36.5 31.539±6 194.0 71.8 1 55.6T20 Undetectable 29.0 6.5 29.7 positive 1 etc; double jlL cell T cell embryo 25±2 45.9 37.1 41.4 Fibroblasts (lung) 29±4 10.3 9,3 10.2 Fibroblasts (synovial fluid) Undetectable 4.8 3.9 4.5 endothelial cells : 7.5 7.9 7.5 Table 13 Inhibition of tumor cell proliferation by 5HT receptor antagonists and vice versa by 5HT conversion effect Tumor cell lines and normal diploid cells were treated with 30 μM ritanserin (1-40 nM concentrations). 5HT2 receptor antagonists with activity against the 5HT2 site in Culture for 48 hours in the presence or absence of 1 μM 5HT. fed. In this case, the addition of 5HT did not affect the control growth treatment. Also, Cultures were performed in triplicate, and the standard error was less than 10% (cells not shown, 3H- TdR uptake Population (cmpxLOOo) control + ritanserin + ritanserin ME180 40.7 2.2 21.4 Jyurukasoto 75.8 0.4 51.6HT29 159.5 25,3 58.40937 68.9 15 ．． 2 45.8M0LT-434,532,855,5BT20 27.3 3 ．． 1 19.1 Normal - normal ≦; partner - wooden plate 1 Fibroblasts 10.3 8.7 8. .

T cell embryo 43.9 40.5 45.5 Fibroblast (synovial fluid) In addition to the agonists and antagonists that can be synthesized scientifically, the above 5 HT The scope of the present invention includes multiple types of mimotobes and/or antibodies directed against two similar receptors. It extends to.

Identification of the novel 5HT2-like receptor allows for the generation of antibodies against the receptor . Furthermore, the antibodies can be used as research probes and for therapeutic purposes. In addition, Techniques for generating such antibodies are well known in the art. Regarding this method, see above. References (Fundament II Immunology, 5th century) 　Editi.

Please refer to Chapter 12 of n). In addition, the antibodies include polyclonal and monoclonal antibodies. Many "types" can occur, including null, chimeric, humanized and humanized. I realized that. Furthermore, the “type” of these antibodies is similar to the novel 5HT2. The receptor has the ability to recognize one or more specific epitopes.

In addition, the identification of the novel 5HT2-like receptor allows us to specifically identify the purified receptor. To identify peptides that bind to proteins, we use a random array of peptide sequences. It can be used as a probe for screening libraries. difference Additionally, antibodies raised against the purified receptor may also be used as probes. Can be done.

For techniques for constructing libraries of peptides in this case, see Quilla (C wirla) Other documents “Bepti)” J (Pept 1des; Proc, N at I, Acad, Sci, USA, Vol, 87.6378-6382, A a vast library of peptides to identify ligands for Please refer to li. For the above screening method, please refer to Devlin ( Devlin) et al. [Random Peptide Library J (Random Pep tide Libraries; 5ience, Vol, 249, 404-40 6.27July 1990) and species of specific protein binding molecules, and Scott et al. Scot, Smith) “Peptide ligand analysis using epitope libraries” Searching for Peptide Ligands ;5science, Vol, 249, 386-390゜27 July 199 Please refer to 0).

References 1. Plaut, M., 1987. Annu, Rev. Immunol, 5:621 2, Hen5on, P. M., 1970. J. Immunol. 105:476 Mechanism of release of composition from rabbit platelets by antigen-antibody complexes and complement ism.

3, Essman, W, B, 1978.5 pect rum, (W, B, Ess man, ed,) New York, p. 15 Serotonin in tissues and body fluids in healthy and diseased bodies.

4.01son, P.S., U.Ljungvist.

and S,-E, Bergentz, 1974. Thromb, Res, 5: 1 Platelet, red blood cell, and fibrin content in experimental arterial and venous thrombin. Quantitative analysis.

5. Sternberg, E.M., H.J., Wender, M., Leun. G, and C, W, Parker, 1987. J. Immunol, 138 :43 Serotonin (5-HT) and other monoamines on mouse macrophages Effect of ``modulation of interferon-gamma-induced phagocytosis.

6. Sternberg, E.M., J.Trtalandl C.W., Par. ker, 1986. J, Tmmunol, 137:276 Effects of serotonin on mouse macrophages: suppression of Ia expression by serotonin and reversal of the inhibitory effect by 5-HT2 serotonergic receptor antagonists.

7, Helstrand, , and S. Hermodsson, 198 7. J. Immunol. 139: 869 Role of serotonin in regulating cytotoxicity of hominid natural killer cells.

8. Choquet, D., and H. Korn, 1988, Proc., Na. t1. Acad, Sci, USA, 85:4557 Two effects of serotonin on voltage-gate conductance in lymphocytes.

9.8onnet, M., G. Lespinats, and C. Burtin , 1984. Ce1l, rmmunol, 83:280 Phytohemagglutinin and antigen-responsive lymphocyte histamine and sero Tonin suppression.

10. Gershorn, R., P. W., Askenase, and M. D, Gershon, 1975゜J, Exp, Med, 142 near 32 Requirement of vasoactive amines for the development of delayed-type hypersensitivity skin reactions.

11, Ame 1sen, J,-C,, R,Meade.

and P, W, Askenase, 1989. J, Immunol, 198 9:3171 A new interpretation of the involvement of serotonin in delayed-type hypersensitivity: the action of two T cells. Inhibition of touch sensitivity by serotonin-2 receptor antagonists.

12. Schmidt, A.W., and S.J., Peroutka, 198 9. Faseb J, 3:225-hydroxytryptamine receptor group.

13, Peroutka, S. J., 1988, Ann.

Rev, Neurosci, 11:455-Hydroxytryptamine receptor parallelism Type.

1.4. Kadan, M.J., A.M., Krohn, M.

J., Evans, R.L., Wa　　tz, and　P.R.

Hartig, J, Neurochem, 43: 601 Rat ago Binding of 1251-lysergic acid diethylamine to serotonin receptors in the brain cortex combination characteristics.

15, Morett 1-Rojas, I, E, G, Ezrai 1son, L., Birnbaumer, M.L.

Entman, and A. J. Garber, 1983, J. Biol, Ch. Em, 258:12499 Skeletal muscle serotonergic and adrenergic activity in rats. Phosphoregulation: II as a probe of sarcolemma receptor function and specificity [125I]diethylamine indolisergic acid and [125I]iodopuin Uses of drawl.

16. Rabinovitch, P.S., C.H., June, A.Gross. man, and J. A. Ledbetter, 1986. J, Immunol , 137:952 Intracellular free calcium response after mitogenic stimulation with PHA or anti-CD3 Heterologous forms of T cells in. Indo-1 and immunofluorescence by flow cytometry simultaneous use of.

17, Ke I Iey, A, 1989. Cytometry, 10 (Print During) Achieve online reagent addition and shorten flow cytometer sample transition times. conditioning of samples.

18. Grynkiewicz, G., M. Poenie and R.Y.T. sien, 1985. J, Biol, Chem, 260:3440 New generation of Ca+10 indicators with significantly improved fluorescence properties.

19, Smlth, C, D, and R, Snyderman, 1987. M methods in Enzymo lgy, 141: 261 human leukemia Guar in receptor-mediated hydrolysis of polyphosphoinositites in spheres Nin nucleotide regulatory protein.

20. Rosoff, P. M., N. Savage and C. A. Dina Rello, 1988. Ce11, 54 Near 3 Diacyl stimulation of interleukin-1 in 1928 bulbs by a novel mechanism Glycerol production.

21, We i ss, A,, R, L, Wiskoc i + and J, 5t obo, 1984. J, Immun.

1133・123 Role of T3 surface molecules in the activity of human T cells: ■ Two species for L-2 production This reflects the situation in which stimulus requests occur during the pre-translation stage.

22, Berridge, M. J., 1987. Ann.

Rev, Biochem, 56:159 inositol triphosphate and Interacting secondary metsengers of two diacylglycerols.

23. Fargin, A., J.R., Raymond.

M.J., Lohse, M., Kob i Ika, M.G.

Caron, and R.J., Leftkowitz.

1988, Nature (London) 335:35 Beta Adrenaline Genomic clone G-21 with similar receptor sequence was found to accept 5-HT1a. Encode the body.

24, Ju I ius, D, , A, B, MacDerm.

tt, R, Axel and T, Jessel, 1988, -3science 241:558 Functional c DNA molecule that encodes the serotonin 1c receptor Characteristic.

25. Pritchett, D. B., A. W. Bach, M. Wozny. O, Tal eb, R, Da I Toso, J, C, 5hin and P , H., Seeburg, 1988. EMBO, J, 7:4135 cloned Structural and functional representation of rat serotonin 5HT2 receptor.

26, DeVivo, M., and S. Maayani, 1986. J.Ph. armacol, Exp, Ther, 238:248 Forskolin-stimulated adenylate in the cerebral hippocampal membranes of Guinean pigs and rats 5HT1a receptor-mediated inhibitory properties of Rasase activity.

27, Conn, P., J6. and E, 5 anders-Bush, 1985 ．． J, Pharm, and Exp, Therap, 234:195 Serotonin-stimulated phosphoinositide turnover/(-2 rats including subcortical Not mediated by 82 binding sites within the cerebral cortex.

28, Conn, P. J., and E. 5anders-Bush, 1984 ．． Neuropharm, 23=993 Inhibits serotonin-stimulated phosphotidylinositol metabolism in the cerebral cortex. A selective 5-HT2 antagonist.

29, Berridge, M.J., and R.F., Irvine, 1989. Nature, 341:197 Inositol Phosphate and Cell Signaling Effects 30 , Zel　ler, J., , E., Weissbarth.

B, Baruth, H, Mielke and H, Deicher, 1983 , Arthritis and Rheumatism, 26:532 Platelet serotonin content in rheumatoid inflammation: correction for clinical activity.

31゜Sternberg, E.M., J.M., HiI　1. G, P, Chr. ousos, T., Kami faris, S. J., Listwak, P. W., G. old, and R, L, Wi Ider, 1989. Proc, Natl, Acad, Sci, IJSA, 86:2374 Chain balls in Lewis rats with joint inflammation. Defects in inflammatory-mediated induction of hypothalamic-pituitary-adrenal axis activity in bacterial cell walls.

32, Baylin, S. B. and Mendes.

hn, G, Endocrine Reviews 1.33, Julius, D,, Live l l l i, T, J,.

Jessel I, T, M, and Axel, R, 5science 244. 1057-1062 (1989) 34゜Julius, D., Huang ,N., ,Livelli, T., J., ,Axel, R., ,and Jesse. I! , T, M, Proc Nat 1. Acad.

Sci, 87. 928-932 35, Buc-Caron, M.H., Lauray.

J. M., Lamblin, D., and Kellermann, O. Pro. c, Nat I, Acad, Sc i, 87. 1922-1926 (19 90) 36, Laubscher, A. and Pletscher, A.J.P. arm, Pharmacol, 31°37, Peroutka, S. J., Ann. , Rev. Neurosci, 11. 45-60 (1988) 38, Sch midt, A.W., and Peroutka, S.J., Fed.Am.So. c, Exp, Bio 1. J, 3. 2242-2249 (1989) 39 , Glennon, R.A., J., Med., Chem.

30, 1-12 (1987) 40, Hamon, M., Bourgoin, S., Hery, S., and Simonnet, G., Mol.

Pharmacol, 14. 99-110 (1978) 41, Koe, B. K., and Weissman.

A, J, Pharmacol, Exp, Sci, 154°42, Aune, T. M,,Kel　Iey,K,A,.

Ranges, G.E., and Bombara, M.

P, J, Immuno 1. (1990) (in press) 43. DeLarco, J. , E., and Todaro, G. J., Proc., Nat. 1. Acad, S c i.

U, S, A, 75.4001-4005 (1978) 44, Todaro, G. , J., ,DeLarco, J.

E., and Fryling, C.M., FASEBJ, 41. 2996-3 003 (1982) 45, Seuwen, in, Magnaldo, 1. .

and Pouyssegur, J., Nature, 335, 254-256. (1988) 46, Seuwen, N., and Pouyssegur, J. , Biochem, Pharmacol, 39°47, Nemecek, G. M., Coughl in.

S, R,, Hand Iey, D, A,, and Moskowi tz, M, A, Proc, Nat 1. Acad.

Sc, 83, 674-678 (1986) 48, B15hop, J.M. Annu, Rev. Biochem, 52. 301-354 (1983) 4 9. Varmus, H.E., Annu, Rev. Genet, 18. 553- 587 (1984) 50, Aune, T. M., and Pogue, S.

L, J, Cl1n, Invest, 84. 863-8751, Finocch iaro, L.M.E., Arzt., E.S., Fernandez-Cast elo.

S., Cr1scuolo, M., Finkielman, S., and Na. hmod, V, E, J, Interferon Res, 8. 705-716 52, Walker, R.F., Friedman, D.

W., and Jimenez, A., Life Sci.

33, 1915-1924 (1983) 53, Altman, A., T. Mustelin and K, M, Coggeshal 1.　1990. Crltical Rev, in Immunol, 10: T lymphocyte activity : Biological models of signal transduction.

54, Puck, J.M., and R.R.Rich.

1984, J. Immunol, 132:1106 to porkweed mitogen. Regulatory interactions governing proliferation of more stimulated T cell subsets.

55, Aune, T. M. and S. L., Pogue.

1989, J. Immunol, 142:3731 CD8+ suppressor T lymph. Occurrence and properties of continuous systems of spheres.

56. Fox, E. J., R. G., Cook, D. E.

Lewis, and R.R. Rich, 1986. J.

Cl1n, Invest, 78:214 Suppressor T cell proliferation signals: In Helper cells stimulated by Hawklide mitogen in vitro are suppressors Produces cell growth factors.

57, Kammer, G. M., 1988. Immunology Today , 9, 222-229 Adenylate cyclase-c A M P-protein kinase.

Pathways and regulation of immune responses.

searr(gu) Time (minutes) Time (minutes) FIG. 4 Time (minutes) FIG. 5 FIG, 6A Ketanserin 6M FIG, 6B FIG. 7 FIG.8 FIG. 77 Added regulatory cells FIG. 12 Ina W Takashi or/’! ! Everyson Iotu, qFIG, 13B FIG. 14 FIG. 15 FIG, 1B IFN7 FIG,’f7A FIG, 1B Assayro number FIG. 20 EG, 21 international search report Fcrm PcT4SA7)10j#mmJ+Item (ill, P &11210W Front page continuation (51) [nt, CL, 5 Identification symbol Internal office reference number A61K 31150 5 ADS 9360-4C39/395 D 9284-4C U　9284-4C I

Claims (15)

[Claims] 1.5, characterized in that it consists of reducing the effectiveness of the HT1a receptor binding site. A method of modulating the function of activated T cells exhibiting 5HT1a receptors. 2. The effectiveness of the 5HT1a receptor binding site is determined by selecting from the group of 5HT ligands. The effectiveness of at least one antagonist can be reduced by administering an effective dose of at least one antagonist. The method according to claim 1, characterized in that: 3. The group of 5HT receptor ligands includes spiperone, mimotope and the 5HT receptor ligand. 3. The method according to claim 2, characterized in that it comprises an antibody against the 1a receptor. 4.5 Functionally reducing the amount of serotonin that can bind to HT1a receptors The function of activated T cells exhibiting 5HT1a receptor characterized by How to adjust. 5. The function of the activated T cells and the activity of the enzyme tryptophan hydroxylase are It is reduced by inhibiting serotonin synthesis by administering an effective dose of an inhibiting compound. 5. The method according to claim 4, characterized in that: 6.5 consists of functionally increasing the availability of the HT1a receptor binding site. Increased regulation of the function of activated T cells exhibiting the characteristic 5HT1a receptor Method. 7. The function of the activated T cells is controlled by 8-hydroxydipropylaminotetralin H. at least one agonist selected from the group of 5HT1a receptor ligands including Br; 7. A method according to claim 6, characterized in that the amount of sterilization is increased by inputting . 8.5HTla receptor by reducing the effectiveness of the 5HT1a receptor binding site. Breastfeeding characterized by consisting of suppressive regulation of the functions of cells presenting the body A method of treating T cell dependence in patients. 9. binding to the 5HT receptor to inhibit the function of the cell. at least one 5HT receptor ligand selected from the group of 5HT receptor ligands. Claim 8, characterized in that the effect is reduced by administering an effective amount of agonist. Method described. 10. The group of 5HT receptor ligands includes spiperone, mimotope and the 5H 10. The method according to claim 9, characterized in that it comprises an antibody to the T receptor. 11.5HT receptors by functionally reducing the amount of serotonin that can bind to them. 5HT1a receptor A method of treating T cell dependence in patients. 12. The proliferation of the activated T cells is controlled by the activity of the enzyme tryptophan hydroxylase. by inhibiting serotonin synthesis by administering effective doses of compounds that inhibit 12. The method according to claim 11, characterized in that: 13. The proliferation of the activated T cells is inhibited by ritanserin, mianserin, spiperone, A group of 5HT receptor ligands comprising mimotopes and antibodies of the 5HT1a receptor. by introducing an effective amount of at least one antagonist selected from 13. The method according to claim 12, characterized in that: 5HT1a receptor receptors including 14.5HT, ritanserin and L-methyl 5HT. Injecting an effective amount of at least one agonist selected from the group of by functionally increasing the effectiveness of the 5HT1a receptor binding site. Because it enhances the function of activated T cells presenting the 5HT1a receptor. immunization in mammals, characterized in that the effective amount is sufficient to enhance cell function. How to treat deficiencies. 15. 5 characterized by comprising an effective amount of serotonin sufficient to enhance cell function. Function of activated T cell subpopulation CD8+ presenting HT1a receptor How to modulate additively.