Regulatory effects of vasoactive intestinal peptide on the migration of mature dendritic cells

The neuropeptide vasoactive intestinal peptide (VIP), released within lymphoid organs from nerve terminals and/or immune cells, plays a significant anti-inflammatory role. It was reported that VIP can induce regulatory dendritic cells (DCs) and promote Th2-type responses. However, the regulatory effect of VIP on the migration and expression of chemokine receptors by DC is mostly unknown. In the present study, we show that VIP exerts a differential effect on the expression of CCR1 and CCR7 by lipopolysaccharide (LPS)-treated mature DCs (mDCs) at both protein and mRNA levels. It up-regulates CCR1 expression but down-regulates CCR7 expression in LPS-stimulated mature DC, thereby differentially regulating the migration of mature DCs in response to CCL5 and CCL19. Our data indicate that VIP functions as a key endogenous anti-inflammatory agent by inhibiting migration of mDCs to draining lymph nodes, thus preventing the induction of an inflammatory immune response.     

Murine T-lymphocytes express vasoactive intestinal peptide receptor 1 (VIP-R1) mRNA

Vasoactive intestinal peptide (VIP), a neuropeptide present in primary and secondary lymphoid organs has been previously reported to inhibit IL-2 and IL-4 production, as well as the proliferation of mitogen- or antigen-stimulated T-cells. Binding studies suggested that the immunoregulatory effects of VIP are mediated through specific VIP-binding sites present on lymphocyte subpopulations. Here we report on the expression of VIP-R1 mRNA in various murine lymphocyte subpopulations. By using RT-PCR, RNase protection assay, cDNA cloning, and sequence analysis, we show that stimulated and unstimulated murine spleen cells, thymocytes, CD4⁺ and CD8⁺ T-cells express VIP-R1. The VIP-R1 fragment amplified from murine brain, thymocytes, spleen cells and CD4⁺ T-cells share identical nucleotide sequences, and a high degree of homology with the corresponding nonlymphoid rat and human VIP-R1 sequences. The expression of VIP-R1 in thymocytes and peripheral lymphocytes, and especially in the CD4⁺ T-cell subset supports the idea that VIP produced or released locally in the lymphoid microenvironment could directly affect cytokine production and proliferation of T-lymphocytes.     

Vasoactive intestinal peptide (VIP) inhibits TGF-beta1 production in murine macrophages

Vasoactive intestinal peptide (VIP) and the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), produced and/or released in the lymphoid microenvironment act primarily as macrophage- and T cell-deactivating agents. In the present study we investigate the effect of VIP and PACAP on the production of TGF-beta1 in the macrophage cell line Raw 264.7 and in peritoneal macrophages. The two neuropeptides do not affect the baseline TGF-beta1 production by unstimulated macrophages, but reduce dramatically TGF-beta1 production by LPS-stimulated macrophages. The effects are mediated through the specific receptors VPAC1, VPAC2, and PAC1. The effect of VIP is mediated primarily through the cAMP pathway, whereas PACAP activates both the cAMP and the protein kinase C pathway. VIP reduces the TGF-beta1 steady-state mRNA levels in both peritoneal macrophages and Raw 264.7 cells treated with LPS. A similar effect is observed upon the in vivo administration of VIP. This report adds VIP and PACAP to the only other neuropeptide, substance P, known to regulate TGF-beta1 production in immune cells.     

The sympathetic control of blood supply is different in the spleen and lymph nodes

OBJECTIVES: The noradrenergic innervation of lymphoid organs controls several immune cell functions and local blood perfusion. Considering that cell and antigen uptake depend on the blood supply to lymphoid organs, the hypothesis was tested that feedback signals from activated immune cells control sympathetic vasomotor activity. METHODS: We determined the blood flow in spleen and mesenteric lymph nodes (mLN) of Wistar Kyoto rats during immune stimulation with endotoxin (LPS; 10 microg/kg) and following disruption of the noradrenergic transmission. RESULTS: Our data indicate that (a) the splenic noradrenaline content, which reflects the density of the sympathetic innervation, is 5 times higher in the spleen than in other peripheral organs and the spleen receives stronger tonic sympathetic input than mLN; (b) immune stimulation with LPS causes a 4-fold increase in the IL-1beta production in the spleen, but only 2-fold in mLN; (c) IL-1beta causes an inhibition of the sympathetic vasoconstrictor tonus in the spleen, but has no significant effect on the noradrenergic vascular tonus in mLN, and (d) in mLN, the local hyperemia induced by LPS is attenuated by the degranulation of vesicular stores of histamine and serotonin, indicating that these monoamines participate in the vasodilator effect of LPS in mLN. CONCLUSIONS: The present experiments, taken together with our previous studies, indicate that the control of blood supply to the spleen and mLN involves different mechanisms. While blood perfusion in the spleen depends on the inhibition of the noradrenergic vasoconstriction by endogenously produced IL-1beta, other vasoactive mediators such as serotonin and histamine play a role in the control of mLN perfusion.     

VIP and PACAP inhibit IL-12 production in LPS-stimulated macrophages. Subsequent effect on IFNgamma synthesis by T cells.

Since IL-12 plays a central role against intracellular pathogens, and contributes to the pathogenesis of immune diseases, its regulation is essential. This study examines the effect of two neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), on interleukin-12 (IL-12) production. VIP/PACAP inhibit IL-12 dose-dependently. Type 1 VIP receptor (VPAC1), and to a lesser degree type 2 VIP receptor (VPAC2), mediate the inhibition of IL-12, primarily through the cAMP/PKA pathway. VIP/PACAP inhibit the production of IL-12, IL-6, tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma) in vivo in endotoxemic mice. The presence of VIP/PACAP in the lymphoid organs and the specific effects on cytokine production offer a physiological basis for their immunomodulatory role in vivo.     

Shedding of membrane-bound CD14 from lipopolysaccharide-stimulated macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide.

Macrophage activation and deactivation play essential roles in the initiation and maintenance of a successful immune response. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), two structurally related neuropeptides, act as macrophage deactivating factors. We reported previously that VIP and PACAP inhibit IL-6, IL-12, TNF alpha and NO production, and enhance IL-10 production, from lipopolysaccharide (LPS)-stimulated macrophages. In this study, we demonstrate that VIP and PACAP down-regulate the expression of CD14, the membrane-bound LPS receptor, by inducing its rapid shedding. The soluble CD14 released by VIP and PACAP corresponds in size to the soluble CD14 released by PMA. Neither VIP/PACAP nor PMA, affect the steady-state levels of CD14 mRNA. The CD14 shedding induced by VIP/PACAP is mediated through the PAC1 specific receptors and the major transduction pathway involves the protein kinase C (PKC). The VIP/PACAP inhibition of TNF alpha and NO occurs through both CD14-dependent and -independent mechanisms, whereas the inhibition of IL-6 production appears to be strictly CD14-dependent. The shedding of CD14 by VIP and PACAP represents an important mechanism by which these neuropeptides limit the macrophage inflammatory response.     

Dual effect of glia maturation factor on astrocytes : Differentiation and release of interleukin-1 like factors

C6 glioma cells, and primary cultures of mouse astrocytes, stimulated with lipopolysaccharide (LPS) release an interleukin-1 like factor (IL-1) which enhances lectin-induced T-lymphocyte proliferation and promotes the release of interleukin-2 (IL-2) by ConA-stimulated thymocytes. In the present study, the glia maturation factor (GMF) was found not only to induce differentiation of glioblasts, but also to elicit the secretion of IL-1 like factors by cultured mouse astrocytes and their precursor cells. GMF was also effective in triggering IL-1 release by macrophages. Contamination of the 23 000 MW GMF preparation with LPS was excluded by the Limulus lysate assay and by using C3H/HeJ LPS-nonresponder mice whose glia and macrophages responded to GMF but not to LPS, by IL-1 release. Through its ability to induce glial differentiation and IL-1 release, GMF may represent an important endogenous signal, triggering both reactive gliosis and the development of an immune response within the central nervous system.     

Murine mucosal T cells have VIP receptors functionally distinct from those on intestinal epithelial cells

Reports suggest that vasoactive intestinal peptide (VIP) binds to lymphocytes and modulates immune responses. The intestines are richly innervated with VIP-producing nerves. Thus, VIP from nerves or other sources may participate in mucosal immunoregulation. To explore this hypothesis further, murine intestinal mucosal inflammatory cells were scrutinized for functional VIP receptors. An [125I]VIP competitive binding assay characterized VIP receptors. Unfractionated lamina propria inflammatory cells bound [125I]VIP specifically. This binding was abrogated by T cell depletion. The VIP receptor on lamina propria T cells was of a single class with a Kd of 9.08 x 10(-9) M. It bound PHI and other peptide analogs poorly. The intestinal epithelial cell had a high-affinity VIP receptor (Kd 4.17 x 10(-10) M) that bound one VIP analog with moderate affinity. Both VIP and ConA stimulated mucosal inflammatory cells to release interleukin-5 (IL-5). Mucosal inflammatory cells depleted of T cells did not release IL-5 in response to VIP or ConA. It is concluded that: (1) some murine mucosal T lymphocytes have VIP receptors that may be distinct from those displayed on mucosal epithelial cells; (2) VIP affects mucosal T lymphocyte function.     

Exposure to alcohol in utero blunts splenic sympathetic neural response to endotoxin and interleukin-1β in the rat

Systemic administration of endotoxin (LPS) or interleukin-1β (IL-1) to prepubertal rats induced a marked increase in splenic but not cardiac norepinephrine (NE) turnover, an index of sympathetic neural activity. In contrast, the splenic neural response was blunted in their fetal alcohol-exposed (FAE) cohorts. Because the sympathetic outflow to lymphoid organs is considered an important immune modulator, the anomalous neural response to immune signals may partly account for the impaired cellular immunity and, thus, for the increased susceptibility to infections associated with FAE.     

Vasoactive intestinal peptide inhibits interleukin (IL)-2 and IL-4 production in murine thymocytes activated via the TCR/CD3 complex

During their development in the thymus, T cells acquire interleukin (IL)-2 and IL-4 inducibility in a developmentally controlled manner. Although the role of IL-2 and IL-4 in T cell development is still unclear, several reports indicated that IL-2/IL-2R and IL-4/IL-4R interactions in the thymus could play an important role in T cell development. The presence of vasoactive intestinal peptide (VIP)-immunoreactive cells and nerve fibers in the thymus suggests the possible local release of the neuropeptide in the thymic microenvironment. VIP has been previously reported to inhibit IL-2 and IL-4 production, as well as the proliferation of mitogen- or antigen-stimulated peripheral T cells. Here we report on the effect of VIP on IL-2 and IL-4 production by and proliferation of murine thymocytes stimulated through the TCR/CD3 receptor. VIP inhibited both IL-2 and IL-4 production, as well as the proliferation of murine thymocytes in a dose-dependent and specific manner. Structurally related peptides such as secretin or glucagon had little or no inhibitory activity. The intact VIP molecule was inquired for the inhibitory effect, since amino- or carboxy-terminal fragments did not inhibit IL-2 production. The inhibitory effect of VIP was observed for VIP additions up to 12 h after the initiation of the cultures, and incubations longer than 3 h were required for maximum inhibitory effects. Through its downregulatory effect on IL-2 and IL-4 production, locally released VIP could potentially effect T cell development within the thymus.     

Vasoactive intestinal peptide can modulate immune and endocrine responses during lipopolysaccharide-induced acute inflammation

OBJECTIVES: In many studies, it has been reported that vasoactive intestinal peptide (VIP) may play an important role in modulation of the immunological response. VIP can be produced by immunological cells, and also the receptors for this neuropeptide are present in many of these cells. The aim of our study was to estimate the effects of the administration of exogenous VIP on serum concentrations of proinflammatory cytokines [interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha] and an anti-inflammatory cytokine (IL-10) during lipopolysaccharide (LPS)-induced acute inflammation. We also estimated the influence of VIP on pituitary [FSH, LH, TSH and prolactin (PRL)], thyroid (T3 and T4), adrenal (corticosterone) and gonadal (testosterone) hormones in response to LPS-induced acute inflammation. METHODS: Male Wistar-Kyoto rats were divided into four groups, which received, respectively, placebo (0.9% NaCl), LPS, VIP and VIP with LPS. The TNF-alpha and IL-6 serum concentrations were measured after 2 h from the time of the administration of the agents, IL-10 was measured after 4 h, and the pituitary, thyroid, adrenal and gonadal hormone concentrations were measured after 2 and 4 h. Cytokine concentrations were estimated using ELISA tests, and hormone concentrations were measured using RIA tests. RESULTS: In our experiments, LPS administration dramatically increased serum proinflammatory cytokine concentrations (TNF-alpha and IL-6) after 2 h and the anti-inflammatory cytokine (IL-10) after 4 h, as well as increasing the serum corticosterone concentration (after 2 and 4 h) and LH (after 2 h). LPS application decreased serum concentrations of T3 and TSH (both after 2 h), testosterone (after 2 and 4 h), FSH after 4 h and PRL after 4 h. VIP administration decreased the serum IL-10 concentration after 4 h and T3 concentration after 2 h and increased serum concentrations of FSH and corticosterone after 4 h. VIP administrated simultaneously with LPS decreased the LPS-induced increase in IL-6 and corticosterone concentrations (consecutively after 2 and 4 h). VIP also enhanced LPS-induced thyroid hormone (T3 and T4) suppression after 4 h and testosterone suppression after 4 h. CONCLUSION: We conclude that VIP can modulate not only immune responses but also hormonal responses during acute inflammation.     

Regulation of the immune system by sympathetic neural mechanisms

The immune system is made up of primary and secondary lymphoid organs, containing lymphocytes and several accessory cell types, which are the key agents of immunological reactivity. Some of the basic features of immune responses are reviewed. Several pathways from the CNS to the immune system are of potential importance in physiological regulation. The sympathetic nervous system innervates all lymphoid organs with noradrenergic fibers. Furthermore, lymphocytes have receptors for sympathetic neurotransmitters, e.g., beta adrenoceptors. Sympathetic denervation by pharmacologic treatment with 6-hydroxydopamine has a marked affect on several immune responses, including antibody production, delayed hypersensitivity, and generation of cytotoxic T lymphocytes. Norepinephrine, epinephrine, and synthetic adrenergic agonists potentiate cytotoxic T lymphocyte responses in vitro. This appears to be mediated via beta 2 adrenoceptors. Studies with adrenoceptor blockers also indicate a possible role for alpha receptors. In sum, our studies indicate that intact noradrenergic innervation is required for normal immune function. Sympathetic neural influence, via norepinephrine release, may be exerted at the cellular (activation, proliferation, secretion of products) as well as the physiologic (antigen localization, lymphocyte migration) levels.     

Differential expression of vasoactive intestinal peptide receptors 1 and 2 (VIP-R1 and VIP-R2) mRNA in murine lymphocytes

Vasoactive intestinal peptide (VIP), a neuropeptide present in the lymphoid microenvironment, modulates cytokine expression and affects T cell proliferation. Recent molecular studies identified two VIP receptors, VIP-R1 and VIP-R2, primarily in nonlymphoid cells. In this study, we investigate the expression of VIP-R1 and VIP-R2 mRNA in unstimulated and stimulated lymphocytes and thymocytes, and in various lymphocyte subpopulations. In contrast to VIP-R1 which is constitutively expressed, the expression of VIP-R2 is induced only following stimulation through the TCR-associated CD3 complex. Both CD4⁺ and CD8⁺ T cells express VIP-R1 and VIP-R2. Two T cell lines, EL-4.IL-2 and D10.G4.1 express exclusively VIP-R2. VIP induces the expression of the VIP-R2 gene in the absence of additional stimuli. Differential expression and regulation of the two VIP receptors in T lymphocytes suggests different physiological roles in mediating the immunomodulatory activities of VIP and related neuropeptides.     

Neuropeptide modulation of the immune response in gut associated lymphoid tissue

The neuropeptides vasoactive intestinal peptide (VIP), substance P, and somatostatin are found in high concentrations in both the central nervous system and the gastrointestinal tract. Specific high affinity receptors for VIP, substance P and somatostatin have been identified on both human and murine lymphocytes, suggesting a role for each of these neuropeptides in a neuroimmune axis. These peptides may be important modulators of mucosal immunity regulating lymphocyte proliferation and trafficking in gut associated lymphoid tissue, synthesis of IgA, and histamine release. Somatostatin antagonism of both VIP and substance P effects has been observed in the immune system. Though the mechanisms by which these neuropeptides modulate immune function have not been completely delineated, current evidence supports the hypothesis that VIP modulates immune function via cAMP dependent pathways while substance P regulation of the immune response involves phospholipid metabolism. Somatostatin inhibition of both cAMP dependent and phospholipid dependent effects has been documented in endocrine tissues. Delineation of the role of these peptide-peptide interactions in modulation of the immune response promises to be a fruitful area for further investigation.     

Induction and regulation of interleukin-6 gene expression in rat astrocytes

Cells that produce interleukin-6 (IL-6) require the presence of signaling molecules since this cytokine is not normally constitutively expressed. It is now established that astrocytes produce IL-6; however, the precise inducing molecules and the kinetics of their action have not yet been clearly identified. In the current study, we show that either interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) exert a strong inducing signal for IL-6 in primary rat astrocytes. When the two cytokines are added together the response is synergistic, suggesting that each cytokine may induce IL-6 gene expression by different pathways. Interferon-gamma (IFN-gamma) does not affect IL-6 expression although if it is added in conjunction with IL-1 beta, an augmented induction of IL-6 occurs. In addition to the cytokines, bacterial lipopolysaccharide (LPS) and the calcium ionophore, A23187, induce IL-6 expression. IL-6 expression can be blocked by the glucocorticoid analogue, dexamethasone. IL-6 induction by LPS/Ca2+ ionophore is more sensitive to the suppressive effects of dexamethasone than is IL-6 induction by TNF-alpha/IL-1 beta. Cycloheximide (CHX), an inhibitor of protein synthesis, markedly increased levels of IL-6 mRNA in both unstimulated and stimulated astrocytes, indicating that ongoing protein synthesis is not required for astrocyte IL-6 gene expression. We propose that astrocyte-produced IL-6 may have a role in augmenting intracerebral immune responses in neurological diseases such as multiple sclerosis (MS), AIDS dementia complex (ADC), and viral infections. These diseases are characterized by infiltration of lymphoid and mononuclear cells into the central nervous system (CNS), and intrathecal production of immunoglobulins. IL-6 may act to promote terminal differentiation of B cells in the CNS, leading to immunoglobulin synthesis.     

VIP Gene Expression in Rat Thymus and Spleen

Vasoactive intestinal peptide (VIP) is a neuropeptide with immunomodulatory properties. In the present study, we demonstrate VIP gene expression in cells of both thymus and spleen in the rat by in situ hybridization. In thymus sections, hybridization signal for VIP mRNA was found in cells in corticomedullary and medulla regions. In the spleen, cells were labeled at the outer area on the periarteriolar lymphoid sheath of the white pulp. Hybridization signal appeared to be in lymphoid cells. These findings suggest that lymphoid cells might produce VIP, which, if released, could exert a paracrine action on central and peripheral lymphoid organs. We suggest that VIP participates in the bidirectional communication between the nervous and the immune systems.     

Role of the cervical lymphatics in the Th2-type hierarchy of CNS immune regulation

CNS immune regulation is intimately dependent on the dynamics of cerebral extracellular fluid circulation. Animal models indicate that following the introduction of antigen into the CNS, normal circulation of interstitial and cerebrospinal fluids provides the opportunity for (a) delivery of CNS-derived antigen to lymphoid organs, as well as, (b) retention of immunologically significant amounts of antigen within the CNS. Thus, even in the absence of disease, CNS-derived antigen can induce antigen-specific activation of naive lymphocytes in lymphoid organs and specific reactivation of lymphoblasts that have migrated into the CNS. The initial peripheral immune response to CNS-derived antigen is induced in cervical lymph nodes and is characterized by a strong antibody response, no delayed-type hypersensitivity, and only priming for cytotoxic T-cell responses. This Th-2 type hierarchy of immune regulation is reinforced within the antigen-stimulated CNS where specific B lymphoblasts are permitted to develop their effector function but cell-mediated immunity is inhibited. Developing a paradigm for CNS immune regulation is important in understanding how CNS disorders in humans are induced, perpetuated, and may be manipulated.     

Expression of tyrosine hydroxylase in lymphocytes and effect of endogenous catecholamines on lymphocyte function

OBJECTIVES: To comprehend the changes and significance of the endogenous catecholamines in the immune system, we explored the synthesis of catecholamines by lymphocytes in various lymphoid organs and in different activated states, and the effect of the endogenous catecholamines synthesized by lymphocytes on the function of the lymphocytes themselves. METHODS: Immunohistochemistry for lymphoid organs (mesenteric lymph nodes, spleen and thymus) and lymphocytes was used to observe their expression of tyrosine hydroxylase (TH), an initial rate-limiting enzyme of the catecholamine synthesis. The contents of catecholamines, including norepinephrine (NE), dopamine (DA) and epinephrine (E), in lymphocytes were tested by means of high-performance liquid chromatography with electrochemical detection. Western blot was used to examine the character and relative quantity of TH-stained protein in lymphocytes, lymph nodes and adrenal medullary tissue. The effect of alpha-methyl-P-tyrosine (alpha-MT), an inhibitor of TH activity, on concanavalin A (Con A)-induced interleukin-2 (IL-2) production was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. RESULTS: TH-positive cells were found in the three examined lymphoid organs, but the lymph nodes had the highest and the thymus had the lowest density. Both TH expression and the contents of NE, DA and E in the Con A-activated lymphocytes were markedly increased in comparison with those in the nonactivated lymphocytes. A band with TH immunoreactivity was seen in the extracts from either Con A-activated lymphocytes or nonactivated cells and the molecular weight of the protein was 59.4 +/- 0.3 kD. However, the relative quantity of the protein was notably higher in the activated lymphocytes than in the nonactivated cells. As a positive control, a similar band of TH immunoreactivity in the adrenal medullary tissue was also obtained. Alpha-MT at the doses of 10(-11), 10(-10) and 10(-9) M was found to significantly facilitate the Con A-induced IL-2 production. CONCLUSIONS: These results suggest that lymphocytes can synthesize catecholamines and their synthesis levels may increase in the activated state, and that endogenous catecholamines synthesized by the lymphocytes can regulate the function of the lymphocytes themselves.     

Pituitary adenylate-cyclase-activating polypeptide expression in the immune system

Although pituitary adenylate-cyclase-activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide with many different immunomodulatory properties, investigations of its source in lymphoid organs are scarce. The present report contributes to the knowledge on the origin and synthesis of this peptide in immune cells of the lymphoid organs and peritoneum using immunohistochemistry, immunocytochemical staining, Western blot and RT-PCR methods. Our study reveals PACAP immunoreactivity in the thymus, spleen and lymph nodes. Cytochemical results show that PACAP is present in thymocytes, lymphocytes and plasma cells from the spleen and lymph nodes. Western blot analysis showed a band corresponding to PACAP for all lymphoid organs studied. mRNA appears in both double (CD4+CD8+)- and single-positive (CD4+CD8-, CD4-CD8+) thymocytes and in T subsets and B cells from the spleen and lymph nodes. In addition, PACAP mRNA is expressed in lymphocytes, but not in macrophages from peritoneal suspensions. Our findings show that PACAP produced by lymphocytes could be added to the growing list of mediators shared by nervous, endocrine and immune systems. Moreover, PACAP could be considered as a lymphocyte-derived cytokine in the central and peripheral lymphoid organs acting on lymphocytes and stromal cells.