The effects of hexose 6-O-sulfate esters on human natural killer cell lytic function

Natural cell-mediated cytotoxicity (NCMC) is inhibited by some neutral hexoses and hexose phosphates at 25 to 100 mM concentrations. In this study we describe the effects of hexose 6-O-sulfate esters on NCMC against K-562 target cells. Mannose 6-sulfate, galactose 6-sulfate, N-acetylglucosamine 6-sulfate, and N-acetylgalactosamine 6-sulfate inhibit NCMC in a dose-dependent manner at concentrations of 10 mM and below. Inhibitory effects of mannose 6-sulfate and galactose 6-sulfate were evident at concentrations as low as 1.25 mM. The neutral forms of these sugars, glucose and glucose 6-sulfate, did not inhibit NCMC over this range of concentrations. Comparison of the inhibitory effects of sulfated and phosphorylated forms of mannose and galactose indicated that the sulfated forms are much more potent inhibitors. Formation of effector cell:target cell conjugates was unaffected by the presence of sugar sulfates. Calcium pulse experiments demonstrated that inhibitory effects of sugar sulfates were exerted after the Ca++-dependent triggering step in the NK lytic process. Kinetic studies showed that addition of sugars as long as 60 min after initiation of cultures yielded potent inhibitory effects. Sugar sulfates were not toxic for effector cell populations and effectors were not refractory for lytic function after removal of sugars. Sugar sulfates were inhibitory against multiple tumor types in both human and murine NK lytic assays. These results suggest that the sugar sulfates inhibit NK cells at a postconjugation, posttriggering step involving lectin-like receptors or lectin-like molecules.     

High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

Vasoactive intestinal peptide (VIP) bound with high affinity (K_d 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His¹]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound ¹²⁵I-VIP whereas VIP(16–28) and VIP(1–22-NH₂) were inactive. The binding of ¹²⁵I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr¹,D-Phe²]GRF(1–29)-NH₂, [pCl-D-Phe⁶,Leu¹⁷]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated ¹²⁵I-VIP was bound to receptors on the cell surface. The internalized ¹²⁵I-VIP was completely degraded to ¹²⁵I-tyrosine which was released from the cells. Degradation of internalized ¹²⁵I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of ¹²⁵I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound ¹²⁵I-VIP, but caused retention of internalized ¹²⁵I-VIP.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Autoantibody to vasoactive intestinal peptide in human circulation

In a radioassay for Vasoactive Intestinal Peptide (VIP)-binding, eight out of 33 plasma samples from healthy human subjects exhibited specific binding ranging from 2.6% to 46.7% of total [125 I]VIP. This binding was competitively displaced by unlabeled VIP. The structurally homologous peptides, Peptide Histidine Isoleucine (PHI) and secretin, were, respectively, 72-fold and 413-fold less potent than VIP in displacing bound [125 I]VIP, whereas the unrelated peptides, neurotensin, eledoisin, bombesin and metenkephalin, were without effect on the binding. The antibody nature of the VIP-binding factor was suggested by its precipitation with ammonium sulfate, attenuation after absorption with Staphylococcus aureus preparations, precipitation with antisera against human IgG and IgM, and coelution with standard IgG and IgM on anion-exchange and high-performance gel-filtration columns. Pepsin treatment of purified IgG fraction yielded a VIP-binding species with apparent molecular weight of 108 +/- 13 kDa that was precipitated by antiserum against the F(ab)2 fragment of the IgG molecule. These results demonstrate the existence in some human plasmas of an autoantibody that binds VIP.     

A natural variant type II G protein-coupled receptor for vasoactive intestinal peptide with altered function

The vasoactive intestinal peptide (VIP) and its G protein-coupled receptors VPAC1 and VPAC2 prominently mediate diverse physiological functions in the neural, endocrine, and immune systems. A deletion variant of mouse VPAC2 has been identified in immune cells that lacks amino acids 367-380 at the carboxyl-terminal end of the seventh transmembrane domain. When expressed at equivalent levels in a human Jurkat T cell line, which has very low endogenous expression of human VPAC1 and VPAC2, wild-type and deletion-variant VPAC2 bound the same amount of 125I-VIP with similar affinity. Unlike wild-type VPAC2, however, deletion-variant VPAC2 did not transduce VIP-elicited increases in intracellular concentration of cyclic AMP, chemotaxis, or suppression of generation of interleukin-2. Natural deletion of part of the last transmembrane domain of VPAC2 thus abrogates signaling functions without apparent alterations of expression or ligand binding.     

High-affinity receptors for vasoactive intestinal peptide on human myeloma cells

Cultured human myeloma cells of the U266 line and leukemic T cells of the Jurkat line bound synthetic [125I]Tyr10-vasoactive intestinal peptide1-28 ([125I]VIP1-28) specifically and with an affinity similar to that of neuroendocrine cells. Specific binding reached equilibrium after 2 h at 22 degrees C for both myeloma cells and T cells, attained a maximum of 57 to 71% of total binding, and was reversed in 1.5 to 3 h by an excess of non-radioactive VIP1-28. Analyses of the ligand concentration-dependence of binding of the ligand concentration-dependence of binding of [125I]VIP1-28 revealed a mean Kd of 7.6 nM for a mean of 41,207 receptors per myeloma cell and 5.2 nM for 12,266 receptors per T cell. The relative affinity of binding of mast cell-derived VIP10-28 free acid and synthetic analogues suggested differences in specificity between lymphocyte and neuroendocrine receptors. Distinct sets of receptors thus appear to mediate the effects of VIP on functions of both antibody-producing cells and T cells.     

High levels of vasoactive intestinal peptide in human milk

The presence of immunoreactive vasoactive intestinal peptide (VIP) in human milk has been demonstrated by high performance liquid chromatography and a specific radioimmunoassay. Immunoreactive VIP-like peptide co-eluted with the synthetic marker on a reversed phase C₁₈ column. The levels of the neuropeptide ranged between 67 and 161 pg VIP/ml milk.     

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.     

Profiling killers; unravelling the pathways of human natural killer cell function

Natural killer (NK) cells are lymphocytes with an innate abilityto recognize and kill infected cells and tumour cells. UnlikeB and T cells, NK cells do not express an antigen receptor.Instead, NK cells detect changes in the phenotype of the targetcell surface; malignant transformation or infection resultingin the loss or gain of particular molecules that are detectedby inhibitory or activating receptors on the NK cell surface.The identification and characterization of NK cells and theirreceptors was made possible by monoclonal antibody technology.The ease with which genes and gene products can now be identifiedand manipulated has accelerated our understanding of NK cellfunction. Furthermore, gene and protein profiling studies arebeginning to refine our understanding of NK cells, their interactionswith other cells and their effector mechanisms. This reviewillustrates some of the basic features of NK cell biology andhighlights the contribution made by post-genomic technologyin defining the molecular mechanisms by which NK cells identifyand kill susceptible targets.      

Glucocorticoids regulate natural killer cell function epigenetically

Although glucocorticoids are well known for their capacity to suppress the immune response, glucocorticoids can also promote immune responsiveness. It was the purpose of this investigation to evaluate the molecular basis for this apparent dichotomous immunologic effect. Glucocorticoid treatment of natural killer cells (NK) was shown to reduce NK cell cytolytic activity by reduction of histone promoter acetylation for perforin and granzyme B, which corresponded with reduced mRNA and protein for each. In contrast, glucocorticoid treatment increased histone acetylation at regulatory regions for interferon gamma and IL-6, as well as chromatin accessibility for each. This increase in histone acetylation was associated with increased proinflammatory cytokine mRNA and protein production upon cellular stimulation. These immunologic effects were evident at the level of the individual cell and demonstrate glucocorticoids to epigenetically reduce NK cell cytolytic activity while at the same time to prime NK cells for proinflammatory cytokine production.     

Ontogeny of vasoactive intestinal peptide in the human fetal digestive tract

Immunocytochemistry and radioimmunoassay were used to assess the appearance time and tissue distribution of vasoactive intestinal peptide (VIP) in the digestive tract of the human fetus. By radioimmunoassay, VIP was measurable from 10 weeks of gestation. The peptide was abundantly distributed in the jejuno-ileum and colon, where the tissue peptide concentration rose from 9-14 weeks of gestation (18.4 +/- 4.4 and 22.0 +/- 5.0 pmol/g wet weight, respectively) to 15-21 weeks (83.0 +/- 21.1 and 98.6 +/- 36.4 pmol/g, respectively). Lower concentrations were recorded in pancreas from 9-14 weeks of gestation (4.3 +/- 0.8 pmol/g) to 15-21 weeks (13.9 +/- 3.7 pmol/g). The peptide concentration was 15.6 +/- 1.9 pmol/g in fundus and 25.5 +/- 3.2 pmol/g in antrum from 15 to 21 weeks of gestation. The highest concentration was recorded in duodenum from 15 to 21 weeks of gestation (118.4 +/- 40.8 pmol/g wet weight). Tissue VIP concentration and age were positively correlated in the jejuno-ileum. By immunofluorescence, immunoreactive VIP was localized in nervous fibers in the muscularis externa, in the submucosa and in the lamina propria. Scarce cell bodies were also found in the myenteric plexus. No immunofluorescent endocrine cells were observed. These results suggest: (1) the early appearance of immunoreactive VIP in gut, as early as 10 weeks of gestation; (2) the peptide, localized in nervous structures only, follows the same distribution pattern as that in adults; (3) the development of VIPergic structures is a continuous process, initiated during the 3rd month of pregnancy.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

The effects of antagonists of vasoactive intestinal peptide on nonadrenergic noncholinergic inhibitory responses in feline airways

Relaxations of the feline intrapulmonary bronchus (IPB) induced by VIP or nonadrenergic noncholinergic (NANC) inhibitory nervous stimulation were unaffected by the VIP receptor antagonist [Ac-Tyr1,D-Phe2]-GRF (1-29) (30 microM). A second VIP antagonist, [pCl-D-Phe6,Leu17]-VIP (30 microM), also had no effect on NANC relaxation responses or IPB sensitivity to VIP. However, responses to three of the four highest VIP concentrations were inhibited by this antagonist. These results indicate that [Ac-Tyr1,D-Phe2]-GRF (1-29) and [pCl-D-Phe6,Leu17]-VIP are not effective competitive antagonists of VIP receptors in feline airways and, hence, have but limited applicability in determining the role of VIP in mediating airway NANC inhibitory responses in this tissue.     

Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide

The circadian system, driven by the suprachiasmatic nucleus (SCN), regulates properties of cardiovascular function. The dysfunction of this timing system can result in cardiac pathology. The neuropeptide vasoactive intestinal peptide (VIP) is crucial for circadian rhythms in a number of biological processes including SCN electrical activity and wheel running behavior. Anatomic evidence indicates that SCN neurons expressing VIP are well positioned to drive circadian regulation of cardiac function through interactions with the autonomic centers. In this study, we tested the hypothesis that loss of VIP would result in circadian deficits in heart rate (HR) and clock gene expression in cardiac tissue. We implanted radiotelemetry devices into VIP-deficient mice and wild-type (WT) controls and continuously recorded HR, body temperature, and cage activity in freely moving mice. Under light-dark conditions, VIP-deficient mice displayed weak rhythms in HR, body temperature, and cage activity, with onsets that were advanced in phase compared with WT mice. Similarly, clock gene expression in cardiac tissue was rhythmic but phase advanced in mutant mice. In constant darkness, the normal circadian rhythms in HR were lost in VIP-deficient mice; however, most mutant mice continued to exhibit circadian rhythms of body temperature with shortened free-running period. The loss of VIP altered, but did not abolish, autonomic regulation of HR. Analysis of the echocardiograms did not find any evidence for a loss of cardiac function in VIP-deficient mice, and the size of the hearts did not differ between genotypes. These results demonstrate that VIP is an important regulator of physiological circadian rhythmicity in the heart.     

自然杀伤细胞和自然杀伤T细胞在动脉粥样硬化形成中的作用

动脉粥样硬化发生发展与免疫细胞参与的免疫反应密切相关,其中自然杀伤细胞主要是通过释放IFN-γ、穿孔素和颗粒酶等方式发挥生物学作用,自然杀伤T细胞通过释放多种细胞因子影响动脉粥样硬化形成,但其具体机制未明。本文就自然杀伤细胞和自然杀伤T细胞对动脉粥样硬化的影响做一综述,为动脉粥样硬化及其相关疾病的防治研究提供新的思路。     

The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs in vitro and in vivo. The VIP/PACAP-generated DCs are CD11c(low)CD45RB(high), do not up-regulate CD80, CD86, and CD40 following LPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptor and protein kinase A, and correlates with the inhibition of IkappaB phosphorylation and of NF-kappaBp65 nuclear translocation. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previously described Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensitivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege.     

Immunomodulation of human natural killer cell cytotoxic function by triazine and carbamate pesticides

Triazine (atrazine) and carbamates (maneb, metiram, and ziram) are used as pesticides on a variety of crops around the world. To our knowledge, there have been no studies dealing with the effects of these compounds on human natural killer (NK) cells cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of atrazine, maneb, metiram, zineb, and ziram on the ability of human NK cells to lyse tumor cells. The compounds were tested in both purified NK cells as well as a cell preparation that contained both T and NK lymphocytes (T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 h to 6 days. Exposure of highly purified NK cells to 10 microM atrazine, maneb, or metiram inhibited K562 tumor cell lysis by 63+/-25, 95+/-4, and 50+/-6%, respectively, after a 24 h exposure and by 83+/-21, 70+/-39, and 48+/-41% after a 6-day exposure. Exposure to 2.5 microM ziram for 24 h caused a 99+/-2% decrease in lytic function and at 1 microM for 6 days caused a 96+/-4% decrease. However, when T/NK cells were exposed to atrazine, maneb, or metiram for 24 h only 10 microM atrazine and maneb caused a significant decreases in lytic function (61+/-13 and 38+/-18%) and after 6 days only atrazine was inhibitory (54+/-12%). A 24-h exposure to 2.5-microM ziram caused a 41+/-51% decrease in function, but a 6-day exposure to 1 microM ziram caused no inhibition of lytic function. The results provide evidence of relative toxic potential for the five compounds and the immunomodulatory effects on both T and NK lymphocyte function.     

Surface-active properties of vasoactive intestinal peptide*

The purpose of this study was to determine whether human vasoactive intestinal peptide (VIP) aggregates in aqueous solution and, if so, whether the peptide interacts with a biomimetic phospholipid monolayer and increases surface pressure. Using a custom-made Teflon trough containing HEPES buffer (pH 7.4) at room temperature and a surface tensiometer, we found that the critical micellar concentration (CMC) of VIP is 0.4 microM. Surface pressure of a dipalmitoylphosphatidylcholine (DPPC) monolayer spread over the HEPES buffer declined significantly over 120 min because of phospholipid decomposition. However, injection of VIP at concentrations above CMC into the subphase of the monolayer elicited a significant concentration-dependent increase in surface pressure that persisted for 120 min (P < 0.05). Unlike VIP, injection of [(8)Arg]-vasopressin at an equimolar concentration only prevented the time-dependent decline in DPPC monolayer surface pressure. Taken together, these data indicate that human VIP aggregates in aqueous solution and expresses surface-active properties at physiological concentrations in vitro. We suggest that these attributes could have a role in modulating the bioactive effects of the peptide in vivo.