NEUROPEPTIDES MODULATE A MURINE MONOCYTE/MACROPHAGE CELL LINE CAPACITY FOR PHAGOCYTOSIS AND KILLING OF LEISHMANIA MAJOR PARASITES

Host-parasite interactions and their outcome constitute a critical and challenging step in disease establishment in cutaneous leishmaniasis. In the present in vitro study we investigated the possible modulating effects of both sensory and autonomic neuropeptides that normally exist in human and mouse skin, on the uptake and leishmanicidal capacity of macrophages on Leishmania (L.) major parasites, using a monocyte/macrophage murine cell line (Raw 264.7). The sensory neuropeptides somatostatin (SOM), calcitonin gene-related peptide (CGRP) and substance P (SP) suppressed the macrophage capacity for phagocytosing L. major promastigotes at different concentrations, 10^-10-10^-5M, however, the suppressive effect of SP does not reach a significant level. CGRP and SP enhanced the leishmanicidal capacity of macrophages at 10^-7M, and 10^-5 M, respectively, whereas SOM was without effect.

The autonomic neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) both suppressed the phagocytic and leishmanicidal capacities of macrophages at various concentrations, 10^-10-10^-5M.

The findings indicate that neuropeptides have modulating effects on macrophage-L. major interactions. These effects might be exerted by a direct action on macrophages or indirectly through induction of other mediators.     

Protecting Effects of Vasoactive Intestinal Polypeptide on Lymphocytes Against Metal Toxicity

The hormonal neuropeptides calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), were investigated for a potential protective effect on thymocytes after a toxic dose of nickel sulfate, giving an inhibition of DNA synthesis. There was a statistically significant increase in the synthesis of DNA from the level caused by nickel sulfate, with VIP, l0^-4-10^-5 mol/l, while the slightly stimulating effects obtained with CGRP, CCK and NPY, were statistically non-significant. This indicates that VIP, at least as pharmacological concentrations, might have protective effects on lymphocytes against metal toxicity.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.     

Mycobacterium vaccae immunization to OVA sensitized pregnant BALB/c mice suppressed placental and postnatal IL-5 and inducing IFN-gamma secretion

Although the development of atopy in the newborn is determined by a multitude of factors, an intense Th1 stimulus early in life could be protective by facilitating a switch away from Th2. Aimed to determine the effect of single Mycobacterium vaccae (M. vaccae) immunization to OVA-sensitized pregnant mice on IL-5 and IFN-γ secretion from placental lymphocytes and splenocytes of offspring.

Pregnant BALB/c mice were divided into 4 groups, OVA-sensitized + M. vaccae immunized, OVA-sensitized, M. vaccae immunized and controls. Sensitization with OVA was initiated before mating, and aerosol OVA challenge were performed during pregnancy. M. vaccae immunization was performed on the 12^th day of pregnancy. IL-5 and IFN-γ levels of placental lymphocytes were analyzed on the 18^th day of pregnancy and splenocytes of offspring on the 2^nd and 28^th days during postnatal period. A single administration of M. vaccae to OVA-sensitized pregnant mice downregulated IL-5 secretion and induced IFN-γ secretion from placental lymphocytes. On the other hand, after M. vaccae immunization downregulation of IL-5 levels and upregulation of IFN-γ secretion persisted in offspring when determined on 2^nd and 28^th days of life. Vaccination with M. Vaccae to OVA-sensitized pregnant BALB/c mice prevented Th2 immune responses by enhancing secretion of IFN-γ and lowering IL-5 levels during pregnancy and the effect persisted during the postnatal period in offspring.     

大鼠、小鼠颌下腺副交感神经节神经肽的免疫组织化学观察

本实验采用免疫组织化学 ABC法对大鼠、小鼠颌下腺副交感神经节细胞的神经肽进行了定性观察。结果证明 :在颌下腺小叶间结缔组织中存在着副交感神经节 ,神经节内神经元胞体呈圆形、椭圆形或不规则形 ;还可看到有发自神经元胞体的细长或短的突起 ,与邻近神经元似有联系。神经元胞体呈 SP、VIP、NPY、SOM和 CGRP免疫反应阳性 ,细胞核为阴性反应。本实验结果提示 :大鼠、小鼠颌下腺副交感神经节细胞内的神经肽可能以神经递质或神经调质的方式 ,调节其复杂的分泌活动     

Peptide-containing nerves in the human pregnant uterine cervix: an immunohistochemical study exploring the effect of RU 486 (mifepristone).

The presence of several neuropeptides (neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), enkephalin (ENK), somatostatin (SOM) was established in the early pregnant human cervix using indirect immunofluorescence immunohistochemistry. Several peptides (VIP, NPY, CGRP, GAL) were present both in free nerves among smooth muscle cells and around blood vessels. Others (SP, SOM) were only seen as single varicosities among smooth muscle cells. Randomized treatment of patients with RU 486 (mifepristone) prior to surgical sampling revealed no clearcut differences in peptide immunoreactivities. After RU 486 treatment, however, there was a tendency towards a decrease of NPY- and VIP-immunoreactivity, and an increase of CGRP-immunoreactivity.     

Peripheral amplification of sweating – a role for calcitonin gene-related peptide

Neuropeptides are the mediators of neurogenic inflammation. Some pain disorders, e.g. complex regional pain syndromes, are characterized by increased neurogenic inflammation and by exaggerated sudomotor function. The aim of this study was to explore whether neuropeptides have a peripheral effect on human sweating. We investigated the effects of different concentrations of calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and substance P (SP) on acetylcholine-induced axon reflex sweating in healthy subjects (total n = 18). All substances were applied via dermal microdialysis. The experiments were done in a parallel setting: ACh alone and ACh combined with CGRP, VIP or SP in various concentrations were applied. Acetylcholine (10⁻² m ) always elicited a sweating response, neuropeptides alone did not. However, CGRP significantly enhanced ACh-induced sweating ( P < 0.01). Post hoc tests revealed that CGRP in physiological concentrations of 10⁻⁷–10⁻⁹ m was most effective. VIP at any concentration had no significant effect on axon reflex sweating. The duration of the sweating response ( P < 0.01), but not the amount of sweat, was reduced by SP. ACh-induced skin blood flow was significantly increased by CGRP ( P < 0.01), but unaltered by VIP and SP. The results indicate that CGRP amplifies axon reflex sweating in human skin.     

A double-label immunohistochemical study of intramural ganglia from the human male urinary bladder neck

Double-label immunocytochemistry was used to investigate the colocalisation of various neuropeptides and the enzymes nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) in intramural ganglia of the human male urinary bladder neck and trigone. Postmortem specimens were obtained from 7 male infants and children ranging in age from 2 mo to 3 y who had died as a result of cot death or accidental trauma. On average 60% of the intramural neurons were non-TH-immunoreactive (-IR) (i.e. presumptive cholinergic) and 40% were TH- and DbβH-IR (i.e. noradrenergic). Within the non-TH-IR population, calcitonin gene-related peptide (CGRP) was found in 65% of cells, neuropeptide Y (NPY) in 90%, nitric oxide synthase (NOS) in 45%, somatostatin (SOM) in 90%, and vasoactive intestinal polypeptide (VIP) in 40%. The corresponding values for the TH-IR neurons were CGRP (54%), NPY (70%), NOS (58%), SOM (73%) and VIP (40%). All the observed bombesin (BOM)-immunoreactivity was colocalised with TH while 90% of VIP and almost all the CGRP was colocalised with NPY. Less than 5% of neurons were immunoreactive for substance P (SP) or met-enkephalin (m-ENK) and some of these also contained TH. Varicose nerve fibres were seen in close proximity to some of the intramural neurons, the majority of such varicosities showing immunoreactivity to CGRP, VIP or TH. Less common were pericellular varicosities immunoreactive to NPY, SOM or SP. These results demonstrate the neurochemical heterogeneity of intramural neurons in the human bladder neck and provide indirect evidence for the complexity of the peripheral innervation of the human urinary bladder.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Protecting effects of vasoactive intestinal polypeptide on lymphocytes against metal toxicity.

The hormonal neuropeptides calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), were investigated for a potential protective effect on thymocytes after a toxic dose of nickel sulfate, giving an inhibition of DNA synthesis. There was a statistically significant increase in the synthesis of DNA from the level caused by nickel sulfate, with VIP, 10(-4)-10(-5) mol/l, while the slightly stimulating effects obtained with CGRP, CCK and NPY, were statistically non-significant. This indicates that VIP, at least as pharmacological concentrations, might have protective effects on lymphocytes against metal toxicity.     

Immunohistochemical characteristics of human paraganglion cells and sensory corpuscles associated with the urinary bladder. A developmental study in the male fetus, neonate and infant

Triple label immunohistochemistry was used to study the coexistence of the catecholamine-synthesising enzymes dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH) and several neuropeptides including neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM) and galanin (GAL) as well as nitric oxide synthase (NOS) in developing pelvic paraganglion cells in a series of human male fetal, neonatal and infant specimens ranging in age from 13 wk of gestation to 3 y postnatal. 13–20 wk old fetal specimens possessed large clusters of paraganglion cells lying lateral to the urinary bladder and prostate gland which were intensely DBH-immunoreactive (-IR) but lacked TH, NOS and the neuropeptides investigated. With increasing fetal age small clusters of paraganglion cells were observed in the muscle coat of the urinary bladder. At 23 wk of gestation occasional paraganglion cells were NOS or NPY-IR while at 26 wk of gestation the majority of paraganglion cells were TH-IR and a few were SOM or GAL-IR. Some postnatal paraganglia within the bladder musculature contained cells which were all VIP, SP or CGRP-IR while others displayed coexistence of NOS and NPY, SP and CGRP, or NPY and VIP. The presence of NOS in certain paraganglion cells indicates their capacity to generate nitric oxide (NO). These results show that human paraganglion cells develop different phenotypes possibly dependent upon their location within the bladder wall. A delicate plexus of branching varicose nerves was observed in the fetal paraganglia which increased in density with increasing gestational age. The majority of these nerves were VIP-IR while others were CGRP, SP, NPY, NOS or GAL-IR. The presence of nerve terminals adjacent to the paraganglion cells implies a neural influence on the functional activity of the paraganglia. Some paraganglia in the late fetal and early postnatal specimens contained Timofeew's sensory corpuscles, resembling pacinian corpuscles in their morphology. The central nerve fibre of these corpuscles displayed immunoreactivity for SP, CGRP and NOS, the latter indicating a possible role for NO in afferent transmission from the urinary bladder. In addition, a few corpuscles were penetrated by a noradrenergic nerve fibre immunoreactive for NPY and TH, which may have a modulatory role on the sensory receptor.     

Inhibition of Natural Killer Activity by Calcitonin Gene-Related Peptide

The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Ba1b/c mice and nude mice was studied. CGRP dose-dependently (10 to 10 M) inhibited NK activity of spleen cells from both strains of mice. This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1. Maximum inhibition by 10^-7 M CGRP was about 60 %. The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Ba1b/c spleen cells. Furthermore, when cells were treated with 10 to 10^-7 M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice. The characteristics of these cells were similiar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM₁ antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin. In addition, the intravenous injection of asialo GM₁ completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM₁ injection. Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration. CGRP may be implicated in the regulation of NK function.     

Effect of sodium fluoride on the murine splenic immune response to Porphyromonas gingivalis in vitro

Spleen cells from saline- and Porphyromonas gingivalis-primed mice were cultured and stimulated with or without P. gingivalis and added with or without various concentration of sodium fluoride (NaF). Cell proliferation, antigen-specific IgG antibodies and both IFN-γ and IL-10 levels were determined by a colorimetric assay, ELISA and commercial ELISA kits respectively. The results showed that NaF at concentration of 1×10^-6 M enhanced but at concentration of 1×10^-1 M abolished the immune response to P. gingivalis, suggesting that NaF at low concentration may act as an adjuvant but at high concentration may be toxic to the P. gingivalis-induced murine splenic immune response in vitro.     

Protecting Effects of Vasoactive Intestinal Polypeptide on Lymphocytes Against Metal Toxicity

Abstract

The hormonal neuropeptides calcitonin gene-related peptide (CGRP), cholecystokinin (CCK), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP), were investigated for a potential protective effect on thymocytes after a toxic dose of nickel sulfate, giving an inhibition of DNA synthesis. There was a statistically significant increase in the synthesis of DNA from the level caused by nickel sulfate, with VIP, l0^-4-10^-5 mol/l, while the slightly stimulating effects obtained with CGRP, CCK and NPY, were statistically non-significant. This indicates that VIP, at least as pharmacological concentrations, might have protective effects on lymphocytes against metal toxicity.     

人胎儿舌下腺副交感神经节细胞内的神经肽

目的：对人胎儿舌下腺组织中神经肽SP、VIP、NPY、SOM和铬粒素A(GGA)的分布特点及共存关系进行免疫组织化学观察。方法：采用HE染色法及免疫组织化学ABC法。结果：在第19w人胚胎舌下腺小叶间结缔组织中，可见有明显的副交感神经节，神经节内神经元胞体分别呈SP、VIP、NPY、SOM和GGA免疫反应阳性，免疫反应强度无明显增龄变化，但神经节内神经元胞体数量随增龄略有增多。结论：SP、VIP、NPY、SOM等神经肽和CGA共存于人胚胎舌下腺副交感神经节神经元内，它们可能调节局部血流量，对腺体的发生和发育有一定作用。     

Pretreatment of Human Peripheral Blood Lymphocytes with Interleukin-2 or Dexamethasone Does Not Alter Their Response to Met-Enkephn in A NK-Cmotoxic Assay

The effect of Met-Enkephalin (MENK; 10^-12 - 10^-8 M) on NK-activity of peripheral blood lymphocytes (PBL) after in vitro treatment (18 h, 37 °C) was examined in 30 young, healthy male donors. In the group as a whole (n = 30), no significant effect of MENK was detected. At the individual level, 18 of 30 donors (60%) responded to MENK either by mild enhancement (up to 8%, 8 responders), or by mild attenuation (up to 12%, 10 responders) of the basal NK-activity. The effect of MENK was donorrelated regarding the dose-response, E/T ratio, and direction of MENK action. The influence of pretreatment of PBL (1 h) with either graded doses of interleukin-2 (IL-2; 3, 25, 50 U/ml) or dexamethasone (Dex; 2.5 × 10^-9, 2.5 × 10^-8, 2.5 × 10^-7 M), on the effect of MENK was also tested. The idea was that pretreatment of PBL would result in predictable, and/or stronger response to MENK. In the group as a whole again no significant effect of MENK was detected on the NK-activity of PBL prestimulated by IL-2 (n = 16), or inhibited by Dex (n = 12). Further, pretreatment of PBL with IL-2/Dex did not significantly alter the intensity of modulation by MENK, which was generally mild. The data obtained have shown that pretreatment of PBL with IL-2 or Dex, regardless of their concentrations, did not significantly alter the frequency of responders to MENK being 50%, 62.5% and 64.3% with 3, 25 or 50 U/ml IL-2, respectively, and 50%, with all concentration of Dex used, as compared to that observed with resting PBL (60%). However, at the individual level physiological concentrations of MENK (10^-12 - 10^-9 M) induced enhancement or/and attenuation of the NK-activity pretreated with IL-2/Dex, respectively. The effect of MENK at the individual level was donor-related regarding the dose-response, E/T ratio, and direction of MENK action. Thus, pretreatment of PBL with graded concentrations of IL-2/Dex did not alter the effect of MENK on NK-activity, regarding the frequency and intensity, as well as the direction of modulation: it remained bidirectional, of low intensity, and independent of the grade of PBL preactivation/inhibition, therefore unpredictable.     

Flavonoid rutin alters the viability and function of mitogen-stimulated splenocytes and thymocytes compared with non stimulated cells

Rutin is a flavonoid obtained from Dimorphandra mollis (Benth.), a medicinal Brazilian plant used as antioxidative, antihemorrhagic, and blood vessel protector. The present study has examined its effects on the viability and function of immune system cells in vitro. Rat spleen and thymus cells were cultured with 10 nM, 1 μM, and 10 μM of the drug in the presence or absence of PWM, LPS, or ConA mitogens. Cellular proliferation was analyzed by H³-thymidin uptake and IFN-γ and IL-10 were measured by ELISA after 48 and 72 hr. Viability was measured by flow cytometry using Annexin V and PI after 24 and 48 hr. The flavonoid rutin inhibited splenocytes and thymocytes proliferation under ConA stimulation observed by an increase on apoptosis levels of thymocytes stimulated with PWM in 24 hr and on splenocytes stimulated with PWM in 48 hr. Function studies showed a decrease on IFN-γ production by splenocytes and thymocytes stimulated with PWM or ConA. Spleen cells cultured with LPS and rutin showed a decrease on apoptosis after 24 hr and an increase on the IL-10 levels after 48 hr. There was no significant variation on the necrosis rate, viability, and function of cells treated with rutin in the absence of mitogenic stimulus.     

大鼠颌下腺颗粒曲管神经肽的分布

目的 研究颌下腺内神经肽的性质、分布。方法 免疫组织化学ＡＢＣ方法。结果 大鼠颌下腺颗粒曲管和纹状管上皮细胞呈ＳＯＭ、ＳＰ、ＶＩＰ和ＮＰＹ免疫反应性,崦腺兆细胞为阴性。结论 大鼠颌下腺导管上皮细胞内含有多种神经肽,可能参与腺体的分泌活动、血液供应乃至消化道功能的调节。     

Chemiluminescence in Human Whole Blood: Modulation by the Cocarcinogens Phorbol Diester and Polychlorobiphenyls

A human whole blood chemiluminescence (CL) assay was established using zymosan as cell activator. Aroclor 1254 was found to inhibit this CL response in a direct linear relation to its concentration, (50% inhibitory dose, (ID₅₀) equal to 5 × 10^-4 M) in diluted blood samples of 10 normal human subjects. In comparison the ID₅₀ of other inhibitors was 1.3 × 10^-3 M for ethylenediamine tetraacetic acid, 3.3 × 10^-3 M for ascorbic acid, 4 × 10^-3 M for reduced glutathione, 1.2 × 10^-3M for ethanol, 2.5 × 10^-1 for methanol and 3.7 × 10^-1 M for dimethyl sulfoxide. Using 12-o-tetradecanoyl-phorbol-13-acetate (TPA) as cell activator the CL response was likewise inhibited by Aroclor 1254 with an ID₅₀ of 4.5 × 10^-4 M. However, it was found that Aroclor 1254 alone has a stimulatory CL effect on otherwise unactivated cells. To compare the mechanisms involved in the CL elicited by the three stimulants zymosan, TPA and Aroclor 1254, the CL signal was measured in the presence of cytochalasin B. Cytochalasin B inhibited zymosan-induced CL, had a smaller inhibitory effect on TPA-induced CL but it could augment the CL response initiated by Aroclor 1254. This pattern of responses implicates Aroclor 1254 in the activation of eicosanoid metabolism as it matches the differential responses reported for arachidonic acid.