Andrzej M. Stanisz

Neuropeptide Regulation of Mucosal Immunity

In this chapter, we discuss in detail our investigations on the potential for neuropeptide regulation of tnucosal immunity. This includes both in vitro and in vivo studies involving substance P (SP), somatostatin (SOM) and vasoactive intestinal peptide (VIP). We have examined the effects of these neuropeptides on lymphocyte proliferation and immunoglobulin synthesis, and on mast cell degranulation. We also present evidence that nerve growth factor (NGE) may be an important molecule in regulating both lymphocytes and mast cells in mucosal tissues. The majority of our studies have involved the gastrointestinal tract and we shall use this organ to illustrate the basic concepts and our hypotheses. It is recognized that not everyone reading this paper will be familiar with the mucosal immune system, nor the potential sources of neuropeptides within mucosae. Accordingly, we have included a brief overview of these areas, with reference to review articles for further information. The reader should note that there is extensive literature on the involvement of other neuropeptides in immune regulation; however, it is not our intention to review this work in detail. Other chapters in this volume present considerable evidence for the extensive interplay of the immune and nervous systems and there is inevitably some overlap between these contributions and our own studies. The reader is referred to the extensive bibliographies throughout this volume for additional useful references.

Effect of immunologic reactions on rat intestinal epithelium: Correlation of increased permeability to chromium 51-labeled ethylenediaminetetraacetic acid and ovalbumin during acute inflammation and anaphylaxis

In these studies we compared jejunal permeability to two probes--chromium 51-labeled ethylenediaminetetraacetic acid (51Cr-EDTA) (mol wt, 360) and ovalbumin (mol wt, 45,000)--under control conditions, during acute intestinal inflammation, and in response to systemic anaphylaxis. Acute inflammation was produced after infection with Nippostrongylus brasiliensis and rats were studied at day 0 (control), day 4 (early), day 10 (acute), and day 35 (postinfection). At the latter stage, immune rats were also studied during anaphylaxis induced by i.v. N. brasiliensis antigen. In each study, blood and urine were sampled over 5 h after the probes were simultaneously injected into ligated loops in anesthetized rats. In controls, small quantities (less than 0.04% and 0.002% of the administered dose for 51Cr-EDTA and ovalbumin, respectively) appeared in the circulation and plateaued at 1 h. During acute inflammation, the appearance of both probes continued to increase with time. Compared with controls, 5-h values for 51Cr-EDTA and ovalbumin were (a) significantly elevated at day 4 (p less than 0.005), (b) increased approximately 20-fold at day 10 (p less than 0.005 and less than 0.01, respectively), and (c) normal at day 35. Urinary recovery of 51Cr-EDTA followed the same pattern. During anaphylaxis, appearance of the probes in the circulation increased at 1 h to values approximately 10-fold those in controls (p less than 0.001 and less than 0.01, for 51Cr-EDTA and ovalbumin, respectively), and then declined. Urinary recovery of 51Cr-EDTA over 5 h was also significantly increased. We conclude that epithelial barrier function becomes impaired during both acute inflammation and anaphylaxis. In this rat model, gut permeability changes to 51Cr-EDTA reflect gut permeability changes to macromolecular antigens. If similar conditions exist in humans, urinary recovery of 51Cr-EDTA may be useful in monitoring intestinal abnormalities associated with inflammation.

Neural Hyperresponsiveness and Nerve Growth Factor in Allergic Rhinitis

Background: In allergic rhinitis, symptoms are triggered not only by allergens but also by environmental irritants. Hereinafter we address the hypothesis that this is reflective of increased responsiveness of the neural apparatus which, in turn, may be attributable to upregulation of nerve growth factor (NGF) in this disease. Methods: We compared subjects with active allergic rhinitis and healthy volunteers in terms of sensitivity and/or magnitude of three nerve–mediated responses, namely (1) the sneezing reflex induced by histamine, (2) the central or nasonasal reflex depicted by contralateral secretions induced by unilateral nasal challenge with capsaicin, and (3) the axonal reflex depicted by plasma extravasation upon capsaicin challenge. We have also measured NGF levels in nasal lavage fluids at baseline and with allergen provocation in rhinitis and healthy subjects. Results: Compared to healthy individuals, subjects with active allergic rhinitis were found to have (1) significantly greater sensitivity and reactivity of the sneezing reflex, (2) significantly greater secretory responsiveness to sensory nerve stimulation, and (3) significantly greater plasma extravasation indicated by albumin leakage following capsaicin nasal challenge. We also found that subjects with active allergic rhinitis have significantly greater baseline levels of NGF in nasal lavage fluids compared to their healthy counterparts, and that these levels can be increased by allergen nasal provocation. Conclusion: The responsiveness of the neural apparatus of the nose is significantly greater in patients with active allergic rhinitis. The increased presence of NGF in the nasal mucosa of these patients supports the hypothesis that this neurotrophin may be implicated in neural hyperresponsiveness.

Interleukin 1β-induced increase in substance P in rat myenteric plexus

Abstract Background: Substance P (SP) is increased in the inflamed intestine of Trichinella spiralis -infected rats, but the underlying mechanism is unknown. Interleukin 1β (IL-1β) messenger RNA and protein is expressed in the longitudinal muscle-myenteric plexus (LM-MP) of this model. Thus, the purpose of the study was to examine the ability of human recombinant IL-1β (hrIL-1β) to increase SP in LM-MP preparations from the intestine of noninfected rats. Methods: LM-MP preparations were incubated with hrIL-1β, and immunoreactive SP (IR-SP) was assessed in the tissues by radioimmunossay or immunohistochemistry. Results: hrIL-1β increased IR-SP in the tissue in a time- and concentration-dependent manner, being maximal after 6 hours at a concentration of 10 ng/mL. The IR-SP could be depleted by scorpion venom, and immunohistochemistry revealed increased staining for SP within nerves of the LM-MP. The action of IL-1β was dependent on protein synthesis, was receptor mediated, and was not due to endotoxin contamination of the cytokine preparation. Conclusions: hrIL-1β stimulates the synthesis of SP in myenteric nerves of rat intestine.

Substance-P-Mediated Intestinal Inflammation: Inhibitory Effects of CP 96,345 and SMS 201-995

The proinflammatory peptide substance P (SP) has been shown to be intimately involved in the local inflammatory processes of Trichinella-spiralis-induced murine intestinal inflammation. Significant increases in SP, increased myeloperoxidase levels coupled with local morphological deterioration of the jejunum and impaired lymphocyte responses to exogenous SP in vitro have been associated with the model. We have recently determined that the elimination of increased levels of SP via anti-SP antibody therapy can spare the murine gastrointestinal tract much of the pathologies associated with the parasitic infection. Here we further demonstrate that the somatostatin analogue SMS 201-995 as well as the SP receptor antagonist CP 96,345 can effectively decrease the inflammation and lost lymphocyte function seen in the jejunum of T. spiralis-infected mice. Again, both intestinal morphology and myeloperoxidase levels were shown to return to normal values upon treatment. The above results suggest that SP is an important modulator of gastrointestinal inflammation.

Nippostrongylus brasiliensis infection evokes neuronal abnormalities and alterations in neurally regulated electrolyte transport in rat jejunum.

Neuronal abnormalities have been described in the intestine of helminth-infected rats. However, the physiological ramifications of these changes have not been determined. Here, we examined epithelial ion secretion, indicated by increases in short-circuit current (Isc), evoked by electrical transmural stimulation (TS) of enteric nerves in Ussing-chambered jejunal tissues from Nippostrongylus brasiliensis-infected rats. Rats were examined at 10 and 35 days post-infection (p.i.); non-infected rats served as controls. TS resulted in significantly reduced ion secretion in jejunum from 10 day p.i. rats compared to controls or jejunum from 35 day p.i. rats. The TS response in tissue from infected rats had, unlike controls, no cholinergic component. Tissues from both non-infected and infected rats were equally responsive to the muscarinic agonist bethanechol, suggesting that the cholinergic defect was neuronal and not an inability of the epithelium to respond to cholinergic stimulation. However, increases in Isc evoked by exogenous substance P (SP) in tissue from rats 10 day p.i. were reduced in magnitude to approximately 25% of control values. Concomitant with these physiological changes, tissue from infected rats contained increased amounts of substance P immunoreactivity and intestinal sections displayed increased numbers of substance P-immunoreactive nerve fibre profiles at both 10 and 35 days p.i. Thus, following N. brasiliensis infection there is a shift in the enteric nervous system away from cholinergic to non-cholinergic regulation, associated with increased amounts of the pro-inflammatory neuropeptide, substance P. We speculate that changes in neuronal structure and function are intimately involved in the co-ordinated multicellular response to intestinal parasitic infection and subsequent gut recovery.

Vagal influences over mast cells

The established microanatomical association of rat intestinal mucosal mast cells (IMMC) and mucosal nerves raises the possibility that there is crosstalk between mast cells and extrinsic nerves that connect to the CNS. The idea of mast cell-CNS interactions is supported by the demonstration that rat mast cell protease II (RMCPII), found predominantly in IMMC, can be conditionally released by pairing an audio-visual cue with antigen challenge. That the vagus nerve is involved in the IMMC-nerve axis was further demonstrated in a series of our studies showing that: (a) vagal afferents penetrate the small intestinal mucosa and contact IMMC; (b) vagotomy causes a reduction in IMMC density, suggesting a trophic relationship (typical of nerve-target interactions); and (c) stimulation of the cervical vagus causes an increase in histamine and serotonin in IMMC. To further investigate the IMMC-nerve axis in a model of post-inflammatory bowel disorders, infection with Nippostrongylus brasiliensis (Nb) was used to demonstrate an increase in mast cell numbers in the intestinal mucosa and mucosal nerve remodelling with hyperinnervation. Administration of Nb antigen resulted in dramatic increases in mesenteric afferent nerve firing in Nb infected rats, that was absent in sham animals. Moreover, challenge of post-Nb rats with 2-methyl-5HT caused increased mesenteric afferent firing, indicating that vagal afferent innervation remains intact in the post-infection state. These data suggest a functional connection between mast cells and extrinsic afferent nerves. Nb infection provides a useful model of altered communication between IMMCs, peripheral nerves and the CNS, as may occur in post-inflammatory disease states. Since a close anatomical relationship has also previously been demonstrated between nerves and IMMC in humans, further understanding the mast cell-nerve axis may be of critical importance in the development of treatments for various human disease states, including functional bowel disorders.

Neuronal Factors Modulating Immunity

In this minireview we will discuss some evidence suggesting that the immune response is under neuronal regulation. In particular, we will concentrate on the effects that various neuropeptides have on immunity both in vitro and in vivo. Of these, vasoactive intestinal peptide, substance P, somatostatin, and calcitonin gene related peptide will be discussed in detail. In addition, the effects of nerve growth factor on the immune system will be presented. Finally, a possible role for these neuropeptides in various diseases and its clinical relevance will be suggested.

The role of vasoactive intestinal peptide and other neuropeptides in the regulation of the immune response in vitro and in vivo

A substantial body of evidence exists to suggest that neuropeptides, including vasoactive intestinal peptide (VIP), can modulate immune responses and that they may play an important role in inflammatory disease states.’ For example, it was shown that VIP is found in increased amounts in the intestinal tissue of patients with Crohn’s disease, an inflammatory bowel disease that is associated with severe immunological dysfunction.’ A similar association between elevated neuropeptide levels and immune abnormalities was found in cystic fibrosis patients.’ Most of our observations on the effects of neuropeptides on immunity have been based on in vitro studies. Vasoactive intestinal peptide in physiological doses inhibited lymphocyte pr~liferation.~” The activity of so called “natural killer” cells is also affected by VIP, although there is some controversy regarding this, possibly due to differences in methodologies used.’*6 Very little is known about their effects in vivo. There is evidence, however, that they may have an immunoregulatory role in vivo. Vasoactive intestinal peptide appears to play an important role in determining the selective migration of murine T lymphocytes into the intestine.‘ Specific receptors for VIP are present on human* and murine lymphocytes? the majority of them on T lymphocytes with an equal distribution among T helper and T suppressor cells. A single class of receptor with dissociation constant in the nanomolar range, which is within the concentration that gives the most pronounced physiological effects, has been described on lymphocytes and lymphoblastic cell lines?” Vasoactive intestinal peptide is said to be present in mucosal mast cells’’ and polymorphonuclear leukocytes I’ as well as in neurons and their endings13 leaving the question of its origin and local synthesis by cells of the immune system as yet unanswered. Vasoactive intestinal peptide can influence immunity in both direct and indirect ways. Its indirect effects include relaxation of vascular smooth muscle and subsequent

Immunomodulatory activities of the somatostatin analogue BIM 23014c: effects on murine lymphocyte proliferation and natural killer activity

We have examined the effect of somatostatin and its octapeptide analogue BIM 23014c on concanavalin A-induced lymphocyte proliferation and target-specific natural killer activity both in vitro and in vivo. Using Peyers patches and spleen as a source of lymphocytes, we found that both peptides modulated immunity in a dose-dependent manner. Comparatively, there was no significant difference between the activity of somatostatin or BIM 23014c in the modulation of immunity. Proliferation, both in vitro and in vivo, was significantly inhibited by both peptides in each organ with a higher specificity towards the Peyers patch lymphocytes. Natural killer activity was also inhibited in both organs in vivo and in vitro. Thus, not only did somatostatin and BIM 23014c have similar effects on proliferation and natural killer activity, but their effect was organ specific. Preliminary data suggest that BIM 23014c works via the same receptor as somatostatin, therefore intimating that these two peptides are both clinically and immunologically similar.