Characterization of the calcitonin gene-related peptide receptor in mouse T lymphocytes

We have identified and characterized specific binding for calcitonin gene-related peptide (CGRP) in mouse T lymphocytes. The binding of 125I-CGRP to mouse lymphocytes was reversible and the rate of dissociation of 125I-CGRP increased with the addition of the guanine triphosphate nucleotide analog, Gpp(NH)p. Saturation experiments, and Scatchard analysis indicated two classes of binding sites for CGRP; the apparent dissociation constants (KD) were 3.5 X 10(-10)M for high affinity binding sites (Bmax, 265 sites/cell) and 4.8 X 10(-8)M for low affinity binding sites (Bmax, 13,000 sites/cell). The KD value for the high affinity binding sites is roughly comparable to the IC50 and ED50 values for inhibition of T lymphocyte proliferation and increase in the cyclic AMP concentration in these cells, respectively. 125I-CGRP bound to mouse T lymphocytes was displaced by unlabeled CGRP with an IC50 value of 3.2 X 10(-10)M; salmon or human calcitonins did not inhibit the specific binding up to 1 X 10(-6)m. Our studies suggest that CGRP may be an important immunoregulatory molecule, and implicate it in the regulation of T cell function.     

Serotonin and immune response: effect of the amine on in vitro proliferation of rat lymphocytes

OBJECTIVE: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells. METHODS: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10(-13) up to 10(-2) M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24-72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6-12 animals and was expressed as a percentage of the control values obtained with mitogen alone. RESULTS: No significant effect of 5HT at concentrations from 10(-13) to 10(-5) M was found. At concentrations of > or =10(-4) M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 x 10(-4) M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells. CONCLUSION: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations.     

Functional assessment and partial characterization of [3H](+)-pentazocine binding sites on cells of the immune system

The existence of sigma receptors on lymphocytes and thymocytes was characterized using [3H](+)-pentazocine. [3H](+)-Pentazocine specifically labels high affinity sigma-type binding sites on T- and B-enriched lymphocyte membranes. The binding is saturable with T lymphocyte sites having a KD value of 401 +/- 85 nM and B lymphocyte sites having a KD value of 302 +/- 46 nM. Likewise, saturable high (KD1 277 +/- 92 nM) and low (KD2 2.5 +/- 1.2 microM) affinity sites for [3H](+)-pentazocine are found on thymocytes as well. In competition studies with lymphocytes, the rank order of potency for competing ligands is (+)-pentazocine = N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2- (1-pyrrolidinyl)ethylamine (BD1008) greater than 1R,2S-(+)-cis-N-(2-(3,4-dichlorophenyl)ethyl]-2- (1-pyrrolidinyl)cyclohexylamine (LR132) greater than or equal to (-)-pentazocine greater than or equal to phenazocine greater than (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulphonate (U-50,488H) = phencyclidine greater than haloperidol = 1,3-di- (o)-tolylguanidine. In competition studies with thymocytes, the rank order of potency for competing ligands is (+)-pentazocine = BD1008 greater than or equal to phenazocine greater than haloperidol greater than 1,3-di-(o)-tolylguanidine greater than phencyclidine greater than (-)-pentazocine. These compounds were also investigated as potential regulatory molecules in mitogen-stimulated lymphocyte proliferation assays. Of the compounds tested, phencyclidine, 1,3-di-(o)-tolylguanidine, haloperidol, and (+)-pentazocine suppress concanavalin A-induced proliferation at high (10(-5) M) concentrations while (-)-pentazocine is inactive. When pokeweed mitogen or lipopolysaccharide are used, these compounds enhance or suppress lymphocyte proliferation depending on the mitogen and concentration of ligand. These results indicate a stereoselective receptor for (+)-pentazocine which is coupled to biological processes of lymphocytes.     

Effect of benzodiazepines on the proliferation of mouse spleen lymphocytes in vitro

Summary The effect of several benzodiazepines (clonazepam, diazepam, Ro 5-4864, Ro 15-1788) and two pineal gland indoleamines (N-acetylserotonin, melatonin) on the spontaneous proliferation of mouse spleen lymphocytes was estimated in vitro by the 3 H-thymidine uptake assay. It was found that diazepam and Ro 5-4864 (a selective peripheral-type benzodiazepine receptor ligand) produced the concentration-dependent inhibition of 3 H-thymidine incorporation into the DNA of these cells. Ro 15-1788, a specific central-type receptor ligand, evoked a slight inhibitory effect in a high concentration (10^–4M), whereas clonazepam did not produce any significant inhibition. When Ro 5-4864 was tested in combination with diazepam, the inhibition of lymphocyte proliferation did not exceed the effect of diazepam given alone. Ro 15-1788 was unable to reverse the inhibitory action of diazepam in the same experimental conditions. Melatonin and its precursor N-actetylserotonin tested in the concentration range of 10^–4–10^–8 M had no significant influence on the spleen lymphocyte DNA replication in our assay system.These data suggest that diazepam inhibition of lymphocyte proliferation is mediated by peripheral-type sites. Additionally, the fact that melatonin and N-acetylserotonin were unable to affect 3 h-thymidine incorporation argues against any benzodiazepine receptor mediated effect of pineal indoleamines on a cellular proliferation.     

Modulation of human lymphocyte proliferation by FLFQPQRFamide, a FMRFamide-like peptide with anti-opiate properties.

The octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8Fa), originally detected in mammalian brain by antisera raised against the invertebrate peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a neuropeptide able to antagonize the actions of both endogenous and exogenous opiates. Since it is well accepted that lymphocytes are targets for opiates, we have tested the effect of F8Fa on T cell proliferation from normal human peripheral blood lymphocytes. Our study shows that F8Fa exerts a concentration-dependent diphasic modulation of human T lymphocyte proliferation. Thus, despite a great variability between individuals, 10(-13) M F8Fa was found to enhance the proliferation of T cells induced by phytohemagglutinin or anti-CD2 monoclonal antibodies, while 10(-7) M F8Fa inhibited T cell proliferation, without affecting cell viability. When F8Fa was tested on monocyte-depleted cell preparations, only the inhibitory effect was observed. These results indicate that F8Fa may stimulate T cells via monocytes, but may also directly inhibit T lymphocyte proliferation. Given the presence of F8Fa-like peptide in human plasma, we suggest that F8Fa may act as a neurohormone in the control of the immune system.     

Immunomodulatory effect of xylazine, an alpha(2) adrenergic agonist, on rat spleen cells in culture

Xylazine is an adrenergic alpha(2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 microM) and inhibitory (50-500 microM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine, another alpha(2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha(2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, enhanced proliferation of splenocytes to Con A, partly abrogated the inhibitory effect of xylazine in the proliferation assay, and, only at high concentration (1000 microM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha(2) adrenoreceptors.     

Effect of endogenous catecholamines in lymphocytes on lymphocyte function

Our previous work has showed that lymphocytes can synthesize catecholamines (CAs). However, role and mechanism of the endogenous CAs in lymphocytes in modulation of immune function are less known. In the present study, we used alpha-methyl-p-tyrosine (alpha-MT), an inhibitor of tyrosine hydroxylase (TH), and pargyline, an inhibitor of monoamine oxydase, to block the synthesis and degradation of CAs in lymphocytes and then observed changes of lymphocyte proliferation induced by concanavalin A (Con A). Phentolamine and propranolol, antagonists respectively to alpha- and beta-adrenoreceptors, were employed to investigate the receptor mechanism. We found that TH mRNA in the Con A-activated lymphocytes was 2.4 times higher in relative density than that in the resting lymphocytes. Similarly, the intracellular and supernatant CAs, including DA, NE and E, of the Con A-stimulated lymphocytes were significantly raised relative to those of the resting cells. alpha-MT (10(-11), 10(-10) and 10(-9) M) facilitated the Con A-induced lymphocyte proliferation, but pargyline (10(-11), 10(-10) and 10(-9) M) attenuated the cell proliferation. Meanwhile, alpha-MT and pargyline respectively led to decrease and increase in the intracellular and supernatant CAs (DA, NE and E) of the Con A-stimulated lymphocytes. Propranolol completely blocked, but phentolamine partly reversed, the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Content of cAMP was remarkably increased in the lymphocytes treated with pargyline alone, but it dropped to control level after these cells were treated with propranolol plus pargyline. These results on the one hand further demonstrate the ability of lymphocytes to synthesize CAs and the enhancive ability of the activated lymphocytes to synthesize CAs, and on the other hand reveal an important role of the endogenous CAs in regulation of function of lymphocytes themselves. Besides, our present findings suggest that CAs synthesized by lymphocytes can secrete out of the lymphocytes via paracrine or autocrine pathway and affect lymphocyte function by beta-adrenoreceptor and cAMP mediating mechanism. Thus, it can be implied that CAs in lymphocytes are also involved in the cross-talk in the neuro-endocrine-immune networks.     

Time-dependent effect of cyclosporine on mitogenic responses and lymphocyte subset populations in rat spleen and submaxillary lymph nodes

Lipopolysaccharide (LPS)- and concanavalin A (ConA)-induced proliferation and T lymphocyte subsets were measured in spleen and submaxillary lymph nodes of male rats injected with cyclosporine (5 mg/kg s.c. for 5 days, at 12.00 or 24.00 h; animals kept under light from 08.00 to 20.00 h daily). One hour before the third injection, Freund's complete adjuvant or its vehicle was administered. A suppressive effect of cyclosporine on the mitogenic action of LPS was seen in the spleen of rats injected with cyclosporine at noon whereas the effect of ConA remained unaffected. CD4+, CD8+ and CD4+-CD8+ cells decreased in spleen and lymph nodes of Freund's adjuvant-injected rats only with cyclosporine given at noon. The results further support occurrence of time-of-day-dependent effects of cyclosporine on lymphocyte subsets and proliferation.     

T-lymphocyte peripheral-type benzodiazepine binding in parkinsonian patients

Human peripheral blood cells, especially lymphocytes and platelets, are being studied increasingly in neuropsychiatric research both as tools for investigating systemic disturbances in neuropsychiatric disorders, and as peripheral models for getting information on central nervous system biochemistry. In this study, we determined T lymphocyte peripheral-type benzodiazepine binding in parkinsonians and controls: significantly reduced B(max) values were found in patients compared with healthy controls, whereas K(d) values were similar. These data argue for an immune response disturbance and a cell energy metabolism impairment in parkinsonian patients, since cell surface peripheral-type benzodiazepine receptors are related with the immune function, and mitochondrial binding sites with energy metabolic pathways.     

Modulation of murine lymphocyte functions by sulfated cholecystokinin octapeptide

The effects in vitro of the sulfated octapeptide form of cholecystokinin (CCK-8) at concentrations ranging from 10(-13) M to 10(-6) M on several functions of murine lymphocytes were studied, i.e. adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis) and spontaneous and phytohemagglutinin (PHA)-mediated proliferation. Lymphocytes were obtained from peritoneal suspension as well as from axillary nodes, spleen and thymus of BALB/c mice. CCK-8, at concentrations from 10(-10) M to 10(-8) M, significantly inhibited the mobility capacity and the PHA-induced proliferation and increased the adherence and the spontaneous proliferation of lymphocytes. A dose-response relationship was observed, with a maximum effect on lymphocyte functions at 10(-10) M. In addition, CCK-8 induced a significant decrease in membrane and cytosol protein kinase C (PKC) activity in murine lymphocytes, as well as an increase of intracellular cyclic AMP levels. These results suggest that CCK-8 is a negative modulator of two important lymphocyte functions in the immune response, i.e. mobility and mitogen-induced proliferation, and that the PKC activity inhibition and cAMP increase could be the mechanisms through which CCK inhibits these lymphocyte activities.     

系统性红斑狼疮患者血清免疫抑制物的初步观察

目的　初步探讨系统性红斑狼疮 (SLE)患者血清中的免疫抑制物质。方法　选用 SLE患者、狼疮肾患者、慢性肾小球肾炎患者血清。以正常志愿者血清为对照。检测血清及其超滤后不同组分对由刀豆蛋白 (ConA)诱导的淋巴细胞转化的影响。结果　与正常对照组相比 ,狼疮性肾炎患者的血清可十分显著地抑制正常淋巴细胞转化 (抑制率在 90 %以上 ) ,超滤后大于相对分子质量为 3 0 0 0 0的组分具有抑制作用 ,而且其抑制强度与不作超滤处理的血清相同。相对分子质量 <3 0 0 0 0的组分未表现出任何抑制作用。临床诊断未明显累及肾脏的 SLE疮患者 ,其血清也具有明显抑制作用 (抑制率在 5 0 %左右 )。慢性肾小球肾炎患者的血清则对正常淋巴细胞转化几乎无抑制作用 (抑制率小于 2 0 % )。结论　 SLE患者血清中可能存在一种能抑制 T淋巴细胞转化的大分子物质 (相对分子质量 >3 0 0 0 0 ) ,它与由神经系统介导生成的应激免疫抑制蛋白的关系值得进一步探讨。     

Methionine-enkephalin stimulates in vitro proliferation of human peripheral lymphocytes via delta-opioid receptors

The influence of methionine-enkephalin (Met-Enk) on in vitro proliferation of human peripheral lymphocytes was investigated. Met-Enk, in the concentration range 10(-12) to 10(-4) M enhanced the proliferative response to suboptimal concentrations of concanavalin A of human peripheral lymphocytes and to cells in the absence of mitogen. The response to Met-Enk in the absence of mitogen was not influenced by the presence of fetal calf serum: similar levels of enhancement were seen in cultures supplemented with 10% autologous serum. Enhancement of proliferation was blocked in a concentration-dependent manner by both naloxone and the delta specific antagonist ICI-174864. The sensitivity to the antagonistic influence of ICI-174864 suggests strongly that the stimulatory influence of Met-Enk on human lymphocyte proliferation in the absence of mitogen is mediated via the delta-opioid receptor.     

Temporal effects of left versus right middle cerebral artery occlusion on spleen lymphocyte subsets and mitogenic response in Wistar rats

The left and right neocortex of the brain has been shown to exert asymmetrical effects on the immune system. In the present study, we used a middle cerebral artery (MCA) occlusion model in Wistar rats to analyze the influence of unilateral CNS ischemia on spleen cell number and function. The occlusion time was 1 h, followed by reperfusion with survival for 0, 2, 7, 14, and 28 days. Changes in plasma norepinephrine levels were used as an index of peripheral sympathetic activity. Results showed that the total number of spleen cells significantly decreased after 2-28 days of survival in animals with cerebral ischemia compared to sham-operated controls. There was no change in the percentage of CD5(+)-CD4(+) T cells, MHC class II(+) cells, or ED1(+) macrophages. However, the percentage of CD5(+)-CD8(+) T cells decreased at 2 days, resulting in an increased CD4/CD8 ratio, and both parameters returned to control levels after 7 days. Mitogen-induced T and B lymphocyte proliferation increased after 0-28 days post-ischemia independently of the mitogen used. There was no difference in immune response or norepinephrine levels between left and right MCA occlusions. These results are consistent with the notion that cerebral ischemia induces mobilization of certain immune cells from the periphery to the brain, where they may contribute to the local inflammatory response. Additionally, the data indicate that cerebral ischemia is followed by a systemic activation of T and B lymphocytes. Absence of asymmetric effects of left versus right stroke, and failure to demonstrate any suppressive effects of left-sided lesions on lymphocyte proliferation, probably reflects the fact that these large cerebral ischemic lesions affect both cortical and subcortical areas.     

Effects of insulin-like growth factors and growth hormone on the in vitro proliferation of T lymphocytes.

The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear cells (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.     

T lymphocytes in cerebrospinal fluid from patients with neurological diseases.

T lymphocytes were determined according to their ability to form rosettes with sheep erythrocytes, in cerebrospinal fluid (CSF) from 120 patients, and in peripheral blood from 59 patients. Normal CSF contained 74.9 +/- 9.6% T lymphocytes. Increased T lymphocyte percentage was found in CSF from patients with active multiple sclerosis (MS), and Guillain-Barré syndrome, as well as the 2 patients with retrobulbar neuritis and the 1 with subacute sclerosing panencephalitis. Patients with stable MS showed no significant change in CSF T lymphocyte percentage. CSF from patients with viral meningo-encephalitis or meningo-radiculitis had a decreased T lymphocyte percentage. This decrease was also found in the 2 patients with malignant disease and the 2 with presenile dementia. An exceptionally low CSF T cell count (5%) was found in 1 patient with cerebellar ataxia.     

Effects of HPA hormones on adapted lymphocyte responsiveness to repeated stress.

Acute stress-induced immune alterations can result in adapted function with prolonged exposure to the same stressor. The present study was designed to evaluate the possible role of the hypothalamic-pituitary-adrenocortical (HPA) axis on the adaptation of spleen lymphocyte responsiveness to repeated stress. For this purpose, we selected a stressful protocol (aversive auditory stimulation) that induced an initial suppression (1 day), followed by a return to control values with repeated application (4 days), of mitogen-induced lymphocyte proliferation. Because rats exposed to 4 days of noise sessions show enhanced adrenocorticotropin (ACTH) and corticosterone levels, we tested the possibility that adaptation of lymphoproliferation by repeated stress was due to a desensitization of splenic lymphocytes to stress-released HPA hormones. The results showed that corticotropin-releasing factor (10(-9) M) and corticosterone (5 x 10(-8) and 10(-7) M), as well as dexamethasone (10(-8), 5 x 10(-8), and 10(-7) M), significantly suppressed lymphoproliferation from both control and stressed rats in a similar way. ACTH (10(-9) and 5 x 10(-9) M) did not significantly influence Concanavalin-A-stimulated spleen lymphocytes. These data indicate that adaptation of lymphocyte proliferation by repeated noise stress occurs without accompanying alterations in lymphocyte responsiveness to HPA hormones.     

骨髓间充质干细胞抑制再生障碍性贫血患者外周血T细胞增殖的实验研究

背景：既往研究发现骨髓间充质干细胞具有免疫抑制作用,而再生障碍性贫血患者存在T淋巴细胞异常活化及增殖。 目的：体外分析骨髓间充质干细胞对再生障碍性贫血患者外周血T淋巴细胞增殖的抑制作用。 方法：分离再生障碍性贫血患者外周血T淋巴细胞,行羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA SE)荧光染色,与体外培养扩增的骨髓间充质干细胞按3︰1比例直接接触法及Transwell法共培养,加入植物血凝素(PHA)刺激T淋巴细胞增殖,并设阴性及阳性对照。流式细胞术检测各组T细胞增殖情况,评价骨髓间充质干细胞对T淋巴细胞增殖的影响。 结果与结论：CFDA SE染色分析显示,直接接触组T细胞增殖较阳性对照组明显减低(P < 0.01);Transwell组T细胞增殖亦较阳性对照组减低(P < 0.05);直接接触组T细胞增殖较Transwell组明显减低(P < 0.01)。骨髓间充质干细胞可能通过直接接触及分泌某些细胞因子方式抑制再生障碍性贫血患者异常增殖的T细胞,以直接接触方式发挥主要作用。     

Stimulation of rat B-lymphocyte proliferation by corticotropin-releasing factor

The mitogenic effect of corticotropin-releasing factor (CRF) on rat lymphocytes was investigated. When rat splenocytes were cultured for 48 hr with CFR, a dose-dependent increase in incorporation of 3H-thymidine (3H-Tdr) was observed, with a maximal response at 10 nM CRF. Comparison of the proliferative effect of CRF on enriched populations of B lymphocytes, T lymphocytes, or macrophages revealed that only B lymphocytes responded following treatment with CRF. When lymphocytes derived from different lymphoid tissues were compared, CRF had a greater proliferative effect on lymphocytes derived from gut-associated lymphoid tissue (mesenteric lymph nodes and Peyer's patches) than on lymphocytes from spleen or inguinal lymph nodes; CRF had no effect on thymocytes. Synthetic fragments of CRF were used to determine which portions of the peptide are recognized by lymphocytes. The C-terminal fragments alpha-helical CRF9-41 and CRF21-41 were as potent as native CRF in stimulating B-lymphocyte proliferation, whereas CRF1-20 did not stimulate proliferation. The activity of these peptides suggests that CRF stimulates lymphocyte proliferation by cellular recognition of structural determinants in the C-terminal one-half of the peptide.     

Comparison of calcitonin gene-related peptide release from rat lymphocytes and dorsal root ganglia neurons

Calcitonin gene-related peptide (CGRP), a neuropeptide contained in primary sensory neurons, has been demonstrated to be synthesized and released by rat lymphocytes in our previous studies. In this study, the release properties and molecular characteristics of CGRP such as immunoreactivity (CGRP-LI) from lymphocytes were compared with those from dorsal root ganglia (DRG) neurons by using CGRP-specific RIA, reverse-phase HPLC, and RT-PCR. Con A and IL-2 could trigger CGRP-LI release from lymphocytes in a time-dependent manner. After 3 days stimulation with 4 microg/ml Con A, the level of CGRP-LI released by lymphocytes was increased from 77.4 +/- 9.6 pg/10(8) cells to 191.1 +/- 13.6 pg/10(8) cells and increased further to 374.5 +/- 38.3 pg/10(8) cells after 5 days. Stimulation with 750 U/ml human IL-2 recombinant (rhIL-2) caused a significantly elevated CGRP-LI release from 75.4 +/- 6.5 pg/10(8) cells to 266.2 +/- 16.2 pg/10(8) cells after 3 days and to 469.1 +/- 43.2 pg/10(8) cells after 5 days. Con A and IL-2 also augmented CGRP mRNA expression in lymphocytes. In the tested period (1-5 days), Con A and rhIL-2 had no stimulating effect on CGRP release from DRG neurons. In contrast, a high concentration of potassium and LPS could induce an acute release of CGRP from DRG neurons, but not from lymphocytes. Lymphocyte-released CGRP-LI was shown to coelute with synthetic rat CGRP (rCGRP) and DRG neuron-released CGRP by reverse-phase HPLC. In addition, to displace (125)I-CGRP from CGRP antibody by lymphocyte-released CGRP-LI was similar to that by synthetic rCGRP. These data suggest that lymphocyte- and nerve-derived CGRP-LI are similar in terms of immunological characteristics, molecular size, and polarity. However, lymphocytes secrete CGRP-LI in response to different stimuli compared to nerve-derived CGRP.     

Suppressive activity of long-term myelin basic protein-specific SJL T cell lines

Myelin basic protein (MBP)-specific T cell lines derived from SJL mice lose the ability to transfer adoptively experimental allergic encephalomyelitis (EAE) after 5-6 restimulations with antigen in vitro. In order to test whether such lines were suppressive, non-encephalitogenic T cell lines were co-cultured with a freshly derived encephalitogenic T cell line. Following co-culture in the presence of MBP and irradiated syngeneic spleen cells the mixture was transferred adoptively to syngeneic recipients. Severe EAE was observed in recipients of the encephalitogenic cell line alone but not in animals which received the co-culture. A co-culture period was required as mixing the encephalitogenic and non-encephalitogenic T cell lines just prior to transfer was without effect. Not all non-encephalitogenic cell lines were found to be suppressive. Culture fluids from the suppressive, but not the non-suppressive lines were found to inhibit MBP-driven proliferation of T cell clones and encephalitogenic lines in vitro. Nineteen of 55 MBP-specific T cell clones derived from suppressive lines were found to elaborate the suppressive supernatant activity. The suppressive effect was not antigen-specific since the same culture supernatants inhibited proliferation of an ovalbumin-specific SJL T cell clone. The suppressive effect became apparent only after T cell lines had lost encephalitogenicity and was not mediated by tumor necrosis factor, lymphotoxin or prostaglandin.