Vasoactive intestinal peptide stimulates T lymphocytes to release IL-5 in murine schistosomiasis mansoni infection.

In murine schistosomiasis, granulomas form around ova deposited in the liver and intestines of infected mice. The granulomas have eosinophils that produce vasoactive intestinal peptide (VIP) and T cells that display VIP receptors. IL-5 is a lymphokine important for the development and maturation of eosinophils. It seemed plausible that VIP, released from eosinophils, may interact with lymphocyte VIP receptors and modulate IL-5 production as part of a feedback regulatory circuit. Thus, we determined whether granuloma T cells make IL-5 and whether VIP modulates IL-5 production. Isolated granuloma cells enriched for T lymphocytes spontaneously released IL-5. Culture of these cells in the presence of VIP increased IL-5 secretion. Spleen cells were also studied. Spleen cells from infected mice did not spontaneously release IL-5 or express IL-5 mRNA and VIP did not stimulate these resting spleen cells to produce this IL. However, these cells did express IL-5 mRNA and secreted IL-5 in response to Con A or soluble egg Ag. VIP could not appreciably modulate IL-5 release when cells were cultured with VIP and the Ag or mitogen. Spleen cells washed free of Con A ceased IL-5 secretion within 24 h. These preactivated splenic T cells resumed vigorous IL-5 secretion in response to either Con A or VIP. Yet only Con A prominently induced IL-5 mRNA expression. VIP was an effective stimulus at concentrations equal to or above the kDa of the VIP receptor on both splenic and granuloma T cells (10(-8) M). It is concluded that, in murine schistosomiasis, VIP invokes IL-5 release from activated T cells that are not undergoing immediate TCR stimulation.     

Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide.

Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated Mr of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the Gs protein-coupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively high expression in the cerebral cortex and hippocampus.     

Modulation of granulomatous hypersensitivity. VI. T lymphocyte subsets influence mast cell density in liver granulomas of Schistosoma mansoni-infected mice

In murine schistosomiasis mansoni, a complex series of cell-cell interactions involving T cell subclasses regulates the intensity of the inflammatory granulomatous response. Recent evidence suggests that granuloma mast cells also participate in the regulatory process by the release of histamine. The current study was performed to determine factors that affect the number of granuloma mast cells. More mast cells were detected in liver granulomas from chronically (20-wk) as opposed to acutely (8-wk) infected mice. Adoptive transfer of spleen cells from 20-wk-infected donors into acutely (6-wk) infected recipients increased granuloma mast cell density. Treatment of spleen cells with anti-Thy-1.2 or anti-Lyt-1.1 antiserum and complement, but not anti-Lyt-2.1 or normal mouse serum, abrogated adoptive transfer-induced, augmentation of granuloma mast cell density. Treatment of acutely infected animals with cyclophosphamide or cimetidine (H2 antagonist enhanced granuloma mast cell density. These data suggest that granuloma mast cell density is dependent upon subsets of T lymphocytes.     

Role of adenylate cyclase in human T-lymphocyte surface antigen capping

Our recent studies indicated that capping of T3, T4 and T8 surface antigens on human T lymphocytes is augmented by interaction of adenosine with a purinergic receptor. We suggested that the T-cell capping process was mediated by an adenylate cyclase-coupled purinergic receptor that resulted in the generation of cAMP and occupancy of cAMP receptors. The present study was undertaken to examine whether activation of adenylate cyclase in the absence of purinergic stimulation is sufficient to regulate surface antigen capping. Treatment of T lymphocytes with forskolin or cholera toxin caused activation of adenylate cyclase and occupancy of intracellular types I and II regulatory subunits of protein kinase by cAMP, as demonstrated by photoaffinity labeling with [8-3H]N3-cAMP. Such treatment augmented the rate of capping of the T3, T4, and T8 antigens, which resulted in a significant decrement in the elapsed time to half-maximal capping of each antigen. These observations support the proposition that the normal T-lymphocyte capping mechanism of both T3+, T4+ (inducer/helper) and T3+, T8+ (suppressor) subsets can be augmented by activation of adenylate cyclase.     

Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone

We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea-pigs, cattle, and pigs all responded potently to bovine (b) PTH-(1-34), whereas considerable between-species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken-cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH-(1-34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI greater than bPTH-(1-34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP-(10-28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, inhibited bPTH-(1-34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH-(1-34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH-(1-34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.     

Distribution of substance P receptors on murine spleen and Peyer's patch T and B cells

Previous studies have demonstrated that the sensory neuropeptide substance P (SP) can modulate immune responses in vitro. Work from this laboratory has shown that SP enhances immunoglobulin synthesis by murine splenic and Peyer's patch lymphocytes stimulated with concanavalin A. One mechanism underlying these effects is the binding of SP to specific receptors on lymphocytes. We examined the distribution of SP receptors on murine T and B lymphocytes and their subsets by one and two color fluorescence-activated cell sorter analysis. The specificity and nature of binding was examined using radiolabeled SP, and competitive inhibition experiments were performed with cold SP. In cytofluorimetry experiments, both T and B lymphocytes from Peyer's patches and spleen were bound to SP, with those from Peyer's patches having a higher proportion than lymphocytes from the spleen. The majority of T cells from both organs bound SP with binding being evenly distributed between Lyt-1+ and Lyt-2+ cells. Similarly, the majority of B lymphocytes from spleen and Peyer's patches showed SP binding. There were no significant isotype-specific differences within any organ. Studies using 125I-labeled SP showed specific binding to all lymphocyte subpopulations examined. SP receptors were fewer in number on cells isolated from spleen than on cells from Peyer's patches although the dissociation constants were similar for all populations examined. These studies demonstrated that SP receptors are present both on murine T and B lymphocytes from Peyer's patches and spleen. There is no evidence for differential SP receptor expression on distinct lymphocyte subsets in spleen or Peyer's patches.     

The effects of vasoactive intestinal peptide on human natural killer cell function

Vasoactive intestinal peptide (VIP) can be found at nerve endings in various tissues and has recently been shown to interact with human lymphocytes through an adenylate cyclase-linked receptor. Because various neuroendocrine factors are thought to influence immune responsiveness, we studied the effect of VIP on natural killer (NK) effector function. Human lymphocytes were incubated with 51Cr-labeled K562 target cells in a 4-hr cytotoxicity assay in the absence or presence of increasing concentrations of VIP. As expected from its activation of adenylate cyclase, VIP was inhibitory at 10(-6) to 10(-10) M. Interestingly, however, when lymphocytes were preincubated with VIP for 30 or 60 min, then washed and added to target cells, a significant augmentation of NK activity ensued. Binding studies revealed that preincubation with VIP resulted in increased numbers of effector-target conjugates, whereas cytotoxic activity in agarose was not affected at the single cell level. Studies with synthetic analogs of VIP revealed that the integrity of the 14-28 C-terminal amino acid sequence was essential for its activity in cytotoxicity. These data strongly suggest a functional role for VIP in modulating immune responses during neuroendocrine interactions with the immune system.     

Properties of vasoactive-intestinal-peptide receptors and beta-adrenoceptors in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.     

Vasoactive intestinal polypeptide and glucagon: stimulation of adenylate cyclase activity via distinct receptors in liver and fat cell membranes

Vasoactive intestinal polypeptide (VIP), a peptide hormone that is chemically and biologically related to glucagon and secretin, stimulates the activity of adenylate cyclase in liver and fat cell membranes. Effects of combinations of VIP with glucagon and secretin at concentrations that maximally activate adenylate cyclase suggest that in adipose tissue, the three hormones act on the same enzyme, whereas in liver, VIP and secretin activate a common enzyme that is distinct from that responding to glucagon. Studies with radioiodinated derivatives of VIP and glucagon indicate that these hormones interact with separate receptors. Secretin, which gives a maximal stimulation of adenylate cyclase activity virtually identical to that elicited by VIP, inhibits the binding of the latter to its receptor. However, the apparent affinity of secretin for adenylate cyclase and for the VIP receptor is about two order of magnitude lower than that of VIP. It is suggested that VIP and secretin may activate adenylate cyclase via a common receptor.     

Cutting edge: vasoactive intestinal peptide acts as a potent suppressor of inflammation in vivo by trans-deactivating chemokine receptors

Chemokines mediate trafficking of leukocytes to sites of inflammation and immune responses through activation of G protein-coupled receptors, which thereby provide appealing targets for novel anti-inflammatory agents. Vasoactive intestinal peptide (VIP) is an immunosuppressive neurotransmitter. We show that VIP inhibited the function of chemokine receptors on monocytes and CD4(+) T lymphocytes, with impaired chemotaxis and calcium flux in response to the cognate chemokine ligands CXCL12, CCL3, CCL4, and CCL5. This was mediated by VIP receptor type 1 and was not caused by chemokine receptor internalization. However, VIP caused dose-dependent phosphorylation of the chemokine receptor CCR5. This trans-deactivation process was studied in a murine model of delayed-type hypersensitivity: continuous infusion of VIP resulted in significant abrogation of monocyte and lymphocyte infiltration. Circulating mononuclear cells from VIP-infused mice were unable to respond to chemokines. VIP may provide a novel approach to treatment of inflammatory diseases through inhibition of chemokine-dependent leukocyte recruitment.     

Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP-binding sites in HT29 cells

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.     

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.     

Homologous and heterologous regulation of the helodermin/vasoactive-intestinal-peptide response in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.     

Substance P but not vasoactive intestinal peptide modulates immunoglobulin secretion in murine schistosomiasis.

Neuropeptides including SP and VIP modulate Ig secretion by in vitro stimulated lymphocyte cultures. It is not known whether these neuropeptides effect the B cell directly, or if they significantly alter humoral immune responses to pathogens. We have previously shown that granulomas derived from schistosome-infected mice contain immunoglobulin secreting B cells (ISC) as well as eosinophils that secrete substance P (SP) and vasoactive intestinal peptide (VIP). It therefore seemed plausible that B cells derived from infected animals might respond to these neuropeptides, and that such responses might effect immunoregulatory signals. In this study, we addressed these issues in the murine Schistosoma mansoni model, at the level of immunoglobulin secretion in single B cells. Spontaneous ISC were observed in both splenic and granuloma cell preparations. The addition of SP resulted in a dose-dependent reduction in the number and size of plaques (a 50% reduction was observed at 10(-9) M). This effect was blocked with SP antagonists. Similar results were observed in T cell-depleted cell cultures. VIP had no effect on ISC number or plaque size. We conclude that SP, but not VIP, decreases spontaneous ISC number and Ig secretion in short-term cultures of spleen and granuloma cells. SP appears to exert its effects at the level of single B cells through a receptor-mediated mechanism and may thus play an immunoregulatory role in schistosomiasis.     

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.     

Peptide T does not affect induction of pineal N-acetyltransferase by vasoactive intestinal peptide

It has been suggested that the HIV virus binds to VIP recognition sites which can be blocked by the octapeptide, peptide T. Stimulation of VIP receptors on pinealocytes activates adenylate cyclase and increases the activity of the enzyme serotonin N-acetyltransferase (NAT). We examined whether peptide T or D-Ala peptide T amide affected this induction. We found no evidence for peptide T interference with NAT induction and conclude that if peptide T inhibits attachment of HIV virus to VIP receptors, it does so at regions other than that occupied by VIP in stimulating adenylate cyclase and NAT.     

Peptide T from human immunodeficiency virus does not interact with VIP receptor-effector system in immunocompetent cells of rat and mouse

Human immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is an appreciable structural similarity between peptide T and an eight amino acid sequence of VIP, it is interesting to investigate the interaction of peptide T with the VIP receptor-effector system of immunocompetent cells from both rat and mouse. In this paper, we show the lack of interaction between peptide T and VIP receptor-effector system in peripheral blood lymphocytes, spleen lymphocytes and macrophages of rat and in macrophages of mouse. These results do not support the hypothesis that HIV through peptide T may interact with the VIP receptor-effector system present in immunocompetent cells.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Modulation of granulomatous hypersensitivity. V. Participation of histamine receptor positive and negative lymphocytes in the granulomatous response of Schistosoma mansoni-infected mice

The possible role of histamine and histamine-receptored inflammatory cells in the granulomatous response of Schistosoma mansoni-infected mice was examined. Special staining revealed the presence of numerous mast cells, many partially degranulated within the liver granulomas. Treatment of infected mice with cimetidine (an H2 receptor antagonist) enhanced, and diphenyhydramine (an H1 receptor antagonist) decreased the granulomatous response. Fluorescein-labeled histamine-rabbit serum albumin conjugate (H-FRSA) and unlabeled conjugate (H-RSA)-coated culture plates were used to identify and isolate cells with histamine receptors. A large proportion of granuloma macrophages, lymphocytes, eosinophils, neutrophils, and splenic lymphocytes had histamine receptors. Elution of adherent cells from H-RSA-coated culture plates with H1 or H2 receptor antagonists suggested that receptors on granuloma cells were predominately H1 with some granuloma lymphocytes bearing H2-type receptors. Splenic lymphocytes from infected mice were functionally divided according to the presence or absence of histamine receptors on their cell surface. Receptor-negative lymphocytes appeared to mediate SEA-stimulated MIF production (TDH cells) and participated in the adoptive transfer of suppression of granulomas (TH cells). Whereas, TS cells appeared to have histamine receptors. Based on these data, it is inferred that lymphocytes that regulate lymphokine production (TS cells) within the granuloma may be triggered via their histamine receptors to exert suppressive activity.     

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.