Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites

Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. This study aimed to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), displayed antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. SP, NPY, VIP and CGRP displayed variable degrees of antimicrobial activity against all the pathogens tested with the exception of S. aureus. These antimicrobial activities add a further dimension to the immunomodulatory roles for neuropeptides in the inflammatory and immune responses.     

Autoimmunoregulation: differential modulation of CD10/neutral endopeptidase 24.11 by tumor necrosis factor and neuropeptides.

Neutral endopeptidase (NEP) 24.11 appears to be an important enzyme in both vertebrate and invertebrate autoimmunoregulation. Activation of human or invertebrate immunocytes that express NEP with substrates such as monokines and neuropeptides results in its increased expression, in other words, upregulation. However, since certain neuropeptides are also substrates for NEP, these activated immunocytes will respond to neuropeptides only at higher concentrations, thus downregulating the response. Specifically, in tumor necrosis factor (TNF)-treated immunocytes, we demonstrate the effects of increased NEP expression on altering the stimulatory activities of the neuropeptides met-enkephalin, melanocyte-stimulating hormone and substance P. We demonstrate the significance of NEP in modulating these responses through the use of specific enzyme inhibitors such as phosphoramidon, thiorphan and captopril. Furthermore, we present evidence suggesting that the individual variations seen in immunocytes from both different and the same donors to activating substances may reflect fluctuating levels of NEP expressed in response to endogenous stimuli. These results indicate that NEP is a highly significant factor in controlling the response(s) of certain immunocytes in man and higher invertebrates to the influence of biologically active substances such as monokines and neuropeptides.     

Localized retinal neuropeptide regulation of macrophage and microglial cell functionality

The functionality of immune cells is manipulated within the ocular microenvironment to protect the sensitive and non-regenerating light-gathering tissue from the collateral damage of inflammation. This is mediated partly by the constitutive presence of immunomodulating neuropeptides. Treating primary resting macrophages with soluble factors produced by the posterior eye induced co-expression of Arginase1 and NOS2. The neuropeptides alpha-melanocyte stimulating hormone and Neuropeptide Y alternatively activated the macrophages to co-express Arginase1 and NOS2 like myeloid suppressor cells. Similar co-expressing cells were found within healthy, but not in wounded retinas. Therefore, the healthy retina regulates macrophage functionality to the benefit of ocular immune privilege.     

Neuropeptide regulation of immediate and delayed hypersensitivity

Peptide mediators of sensory nerves that are released in tissues by noxious stimuli or inflammatory reactions rapidly elicit local and systemic responses similar to those of immediate hypersensitivity. These sensory neuropeptides affect functions of smooth muscles, blood vessels, leukocytes, and epithelial glands both directly and indirectly, through the actions of mediators released from mast cells stimulated by the peptides. Stereospecific receptors transduce the effects of neuropeptides of the peripheral nervous system (PNS) and central nervous system (CNS) on diverse functions of human, murine and guinea pig mononuclear and polymorphonuclear leukocytes, mast cells, and basophils in vitro and in vivo. Stimulatory and inhibitory effects of neuropeptides on leukocytes are attained in vitro at concentrations which are similar to those in the circulation and in tissues. The dissociation constant (KD) for the binding of a neuropeptide to its leukocyte receptor is within the range of concentrations that evoke cellular responses critical to immunity and hypersensitivity. Neuropeptides exhibit both cellular and stimulus specificities, as exemplified by the greater potency of substance P in activating mucosal than connective tissue mast cells and the capacity of somatostatin to inhibit the release of mediators from basophils challenged by IgE-dependent mechanisms, but not by basic peptides or ionophores. The selective release of distinct neuropeptides from different subsets of sensory nerve endings, the specificity of neuropeptide recognition by mast cells, basophils, lymphocytes, and other target cells, and the diversity of relevant activities of the neuropeptides suggest that the nervous system may initiate and modulate immediate and delayed hypersensitivity by unique mechanisms.     

Somatostatin and substance P induced in vivo by lipopolysaccharide and in peritoneal macrophages stimulated with lipopolysaccharide or interferon-gamma have differential effects on murine cytokine production

We have investigated whether lipopolysaccharide (LPS) induces substance P (SP) and somatostatin (SOM) in popliteal lymph nodes in vivo and whether macrophages are a source of SP and SOM in vitro. We have also investigated the effect of SP and SOM treatment on the production of cytokines. SP reached a maximum 3 days after injection of LPS (100 microg/footpad) and then declined. SOM expression after LPS injection reached a maximum at 5-7 days. Stimulation of thioglycolate-elicited peritoneal macrophages with LPS (20 microg/ml), recombinant interferon-gamma (rIFN-gamma, 100 U/ml), and LPS plus rIFN-gamma induced SOM and SP. Thioglycolate-elicited, unstimulated peritoneal macrophages also synthesized these peptides. SOM (10(-12)-10(-8) M) significantly inhibited IL-6 and IFN-gamma production, whereas SP at those concentrations enhanced cytokine production by activated lymphocytes and macrophages. These findings suggest that neuropeptides which originate from macrophages and nerve fibers act as immunomodulators to mediate changes in the pattern of cytokine production.     

In vitro and in vivo effects of tachykinins on immune cell function in guinea pig airways

The effects of sensory neuropeptides substance P (SP) and neurokinin A (NKA) on immune cell recruitment and macrophage activation were determined. Guinea pigs exposed to capsaicin aerosol exhibited eosinophil and neutrophil influx into their bronchoalveolar lavage (BAL) fluid 24 h after treatment; SP aerosol elicited eosinophil influx, whereas NKA aerosol exposure caused neutrophil recruitment. Inhalation of capsaicin, NKA or SP aerosols also enhanced superoxide production induced by zymosan in cultured alveolar macrophages. Incubation of alveolar macrophages with SP or NKA in culture for the same time (24 h) did not potentiate the response to zymosan. Hence, tachykinin-mediated airway effects may not be the result of direct actions on target cells but rather involve alternate mechanisms and mediators which do not necessarily reflect in vitro data.     

Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P

The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. Recently, the involvement of neuropeptides has been demonstrated to play an important role in the inflammatory cascade. Neuropeptides such as Substance P are released by cutaneous neurons and modulate the function of immunocompetent and inflammatory cells as well as epithelial and endothelial cells. Substance P has been shown to function as a mediator for cell proliferation, cytokine production, and as an upregulator of various cell surface receptors. In this study, we show the effect of Substance P on the production of Tumor Necrosis Factor-alpha, Interleukin 1-beta, Interleukin 2, and Interleukin 6 by T-lymphocytes, macrophages and neutrophils. These data demonstrate that pathophysiological levels of Substance P induce production of cytokines in all three cell populations tested. Interestingly, T-cells demonstrated the highest percentage of cells expressing all four cytokines. In contrast, macrophages and neutrophils produced the highest absolute levels of cytokines. The elucidation of mediating mechanisms of Substance P activation of leukocytes is crucial to the understanding of the cutaneous inflammatory cascade and involvement of the peripheral nervous system on the immune system. These findings suggest that Substance P participates in the complex network of mediators that regulate cutaneous inflammation and potentially the rate of wound healing.     

Receptor–receptor interactions in central cardiovascular regulation. Focus on neuropeptide/α<Subscript>2</Subscript>-adrenoreceptor interactions in the nucleus tractus solitarius

Summary. The nucleus tractus solitarii (NTS) is a key nucleus in central cardiovascular control. In this mechanism it is well known the role of the α₂-adrenoreceptors for the modulation of the autonomic pathways. Moreover a number of neuropeptides described in the NTS, including Neuropeptide Y (NPY), Galanin (GAL) and Angiotensin II (Ang II), have different roles in regulating the cardiovascular function within this nucleus. We show in this review several data which help to understand how these neuropeptides (NPY, GAL and Ang II) could modulate the cardiovascular responses mediated through α₂-adrenoreceptors in the NTS. Also we show for the first time the interactions between neuropeptides in the brain, specifically the interactions between NPY, GAL, and Ang II, and its functional relevance for central cardiovascular regulation. These data strength the role of neuropeptides on central autonomic control and provide some evidences to understand the neurochemical mechanisms involved in the cardiovascular responses from the NTS.     

Stimulation of phagocytic function in mouse macrophages by neurotensin and neuromedin N

The neuropeptides neurotensin and neuromedin N (from 10⁻¹² M to 10⁻⁹ M) have been showed in this study to stimulate significantly in vitro several steps of the phagocytic process: adherence to substrate, chemotaxis, ingestion of inert particles (latex beads) and production of superoxide anion measured by nitroblue tetrazolium reduction in resting peritoneal macrophages from BALB/c mice. A dose-response relationship was observed, with a maximal stimulation of the phagocytic process at 10⁻¹¹ M. The two neuropeptides induced no change of intracellular cyclic AMP in murine macrophages. Moreover, adherence and chemotaxis decreased significantly in the presence of EGTA (1 mM), a chelator of extracellular Ca²⁺, or ryanodine (0.5 mM), a blocker of a Ca²⁺-gated channel from the endoplasmic reticulum, in both controls and samples with the addition of neurotensin or neuromedin N. These results suggest that there is no relation between the cAMP messenger system and the phagocytic process stimulation in murine peritoneal macrophages by neurotensin or neuromedin N. In addition, the results observed with EGTA and ryanodine could indicate that these two neuropeptides produce their effects through an increase of intracellular Ca²⁺ concentration.     

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.     

Characterization of neuropeptides extracted from canine intestine

Quantitative determination of neuropeptides in biologic tissues by radioimmunoassay requires both an efficient extraction of neuropeptides as well as maintenance of immunochemical reactivity. Vasoactive intestinal peptide, substance P, and met⁵-enkephalin were chosen for this study because they are neuropeptides which appear to be involved in multiple physiologic systems. Since all three neuropeptides have a methionine residue within their amino acid sequence, oxidation of methionine to methionine-sulfoxide during the extraction process could diminish their immunochemical reactivity. Multiple factors that might be important in extracting these neuropeptides from canine intestine, including pH of the solvent, tissue homogenization, heating, and addition of enzyme inhibitors, were examined. Concentrations of vasoactive intestinal peptide-like immunoreactivity and substance P-like immunoreactivity were significantly higher in acidic solvents, and tissue homogenization appeared to increase the concentrations of these two neuropeptides. Substance P-like immunoreactivity was increased by heating after tissue homogenization, suggesting heat-induced denaturation of tissue enzymes liberated by homogenization. Separation of acidic tissue extracts by high performance liquid chromatography followed by radioimmunoassay for all three neuropeptides revealed minor acid-induced oxidation of substance P. These results should be useful for planning the extraction of these three neuropeptides from other tissues.     

The neuro-B cell link of peptidergic innervation in the Bursa Fabricii.

The Bursa Fabricii, restricted to birds, specifically provides the microenvironment for B-cell maturation. The presence of nerve fibers containing immunopotent neuropeptides in immune organs opens interesting perspectives on the understanding of neuroimmune communication. As an organ for the development of only B-lymphocytes is not known in mammals, the contribution of a peptidergic innervation to the microenvironment of B-cells is not known. Therefore, we studied the peptidergic innervation of the Bursa Fabricii as an organ of B-cell maturation. Four different neuropeptides were found in nerve fibers of the Bursa Fabricii: tachykinins (TK), vasoactive intestinal peptide (VIP), galanin (GAL), and calcitonin gene-related peptide (CGRP). All these neuropeptides were distributed throughout the different bursal compartments, with the exception of the medulla of the bursal follicles, where the early stages of lymphocyte maturation are found. Double immunofluorescent studies revealed three different fiber populations: One containing coexisting VIP and GAL, the second TK and CGRP, and the last TK and no CGRP. Some of the TK-ir fibers may contain coexisting VIP. As demonstrated by double labeling for neuropeptides and lymphocytes, these heterologous fiber populations were found adjacent to B-cells in the follicle cortex. In addition, VIP-ir fibers were seen in association with macrophages. The origins of the fiber populations and the possible functional implications of our findings are discussed.     

Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypeptide inhibit the MEKK1/MEK4/JNK signaling pathway in LPS-stimulated macrophages

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, downregulate TNFalpha expression in LPS-stimulated peritoneal macrophages and Raw 264.7 cells. We showed previously that VIP/PACAP change the composition of the CRE-binding complex in the TNFalpha promoter from highc-Jun/(low)CREB, characteristic for LPS-stimulated macrophages, to lowc-Jun/(high)CREB, characteristic for the unstimulated cells. In the present study we examined the effects of VIP/PACAP on the MEKK1/MEK4/JNK transduction pathway, and on the subsequent changes in Jun family members. Our studies indicate that VIP/PACAP inhibit MEKK1 activity, and the subsequent phosphorylation of MEK4, JNK, and c-Jun. Treatment with VIP or PACAP results in a decrease in AP-1 binding, and a marked change in the composition of the AP-1 complexes from c-Jun/c-Fos to JunB/c-Fos. Western blots confirm that VIP stimulates JunB production in LPS-stimulated macrophages. Both the inhibition of the MEKK1/MEK4/JNK pathway, leading to the reduction in phosphorylated c-Jun, and the stimulation of JunB, are mediated through the specific VPAC1 receptor and the cAMP/PKA pathway. The VIP/PACAP interference with the stress-induced SAPK/JNK pathway in stimulated macrophages may represent a significant element in the regulation of the inflammatory response by the endogenous neuropeptides.     

Targeted Disruption of the Galanin Gene Attenuates Inflammatory Responses in Murine Skin

The release of neuropeptides from primary sensory nerve fibers has been implicated in the modulation of local immune responses in surface tissues, such as the skin and the gastrointestinal mucosa, thereby inducing neurogenic inflammation, which is characterized by plasma extravasation and vasodilatation. In addition, cytokines, either alone or in conjunction with neuropeptides, initiate recruitment of immunocompetent cells such as neutrophils during the initial phases of inflammation. Growing evidence suggests that the neuropeptide galanin plays an important role in skin immune defense and pathophysiology. In this paper, we report that adult mice carrying a loss-of-function mutation in the galanin gene (galanin knockout, Gal KO) demonstrate an absence of the normal neurogenic inflammatory response, upon treatment of the skin either with the vanilloid receptor 1 agonist capsaicin or noxious heat. Furthermore, a lack of an acute inflammatory edema induced by coinjection of substance P and calcitonin gene-related peptide was observed. In addition, Gal KO animals also exhibit a deficit in neutrophil accumulation in the skin after exposure to noxious heat, carrageenin, or tumor necrosis factor alpha. These data indicate that Gal KO mice demonstrate abnormal neurogenic inflammatory responses in murine skin compared to strain-matched wild-type mice.     

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.     

Differential responses of hypothalamic cAMP and cGMP to substance P and neurotensin in ovariectomized rats

Hypothalamic cAMP and cGMP levels in ovariectomized rats were evaluated after intravenous (IV) pulse injection and/or intraventricular (IVT) injection of substance P and/or neurotensin. Intravenous substance P lowered hypothalamic cAMP concentration whereas IVT injection of 2.5 micrograms substance P produced significant increase in cAMP levels. On the other hand, IV administration of neurotensin failed to alter hypothalamic cAMP levels while IVT injection induced significant decrease in cAMP. Intravenous pulse injection of substance P elevated hypothalamic cGMP levels while IVT injection decreased cGMP concentration. Hypothalamic cGMP concentration was not modified by IV administration of neurotensin. However, IVT injection of neurotensin significantly elevated cGMP levels. Since a number of neurotransmitters/neuropeptides exert their action through cyclic nucleotides the present results indicate differential responses of cAMP and cGMP to substance P and neurotensin and implicate a mediatory role for cAMP and cGMP in the neuroendocrine action of substance P and neurotensin.     

Neurotransmitter/neuropeptide interactions in the regulation of neurohypophyseal hormone release

Regulation of neurohypophyseal hormone release reflects the convergence of a large number of afferent pathways on the vasopressin (VP)- and oxytocin-producing neurons. These pathways utilize a broad range of neurotransmitters and neuropeptides. In this review, the mechanisms by which this information is coordinated into appropriate physiological responses is discussed with a focus on the responses to agents that are coreleased from A1 catecholamine nerve terminals in the supraoptic nucleus. The A1 pathway transmits hemodynamic information to the vasopressin neurons by releasing several neuroactive agents including ATP, norepinephrine, neuropeptide Y, and substance P. These substances stimulate VP release from explants of the hypothalamo-neurohypophyseal system and certain combinations of these agents elicit potent but selective synergism. Evaluation of the signal cascades elicited by these agents provides insights into mechanisms underlying these synergistic interactions and suggests mechanisms responsible for coordinated responses of the VP neurons to activation of a range of ion-gated ion channel and G-protein-coupled receptors.