Ron H. Stead

Mast cells are closely apposed to nerves in the human gastrointestinal mucosa

Mast cell/nerve associations have been recorded in several publications; however, the human gastrointestinal tract has received little attention. Accordingly, mucosal samples from small bowel, appendix, and large bowel were studied. Combined histochemical/immunocytochemical techniques revealed that the proportion of mast cells apposed to nerves ranged from 47.08% +/- 6.10% to 77.66% +/- 4.26%. The highest incidence of contact was observed in the appendix; where the apparent nerve density was also greater than in the large or small bowel. Electron-microscopic studies revealed many mast cells adjacent to nerve fibers and membrane-to-membrane contact between axonlike processes and mast cells. Often, these processes were dilated, as were axons in adjacent nerve fibers. These data provide a microanatomic basis for potential communication between nerves and mast cells in the human gastrointestinal mucosa. This may be of physiologic significance in the normal individual and important in disease processes, such as inflammation and fibrosis.

The Role of Mast Cells in Inflammatory Processes: Evidence for Nerve/Mast Cell Interactions

In the rat, there is considerable evidence of mast cell/nerve interaction both in the normal and infected intestine. Between 67 and 87% of all mast cells in the intestinal lamina propria of rats infected 22-35 days earlier with Nippostrongylus brasiliensis were touching nerves. These membrane contacts were between subepithelial mast cells and nonmyelinated nerves containing substance P, calcitonin gene-related peptide and neurone specific enolase. 2.5S nerve growth factor (NGF) has a significant enhancement effect on antigen-induced histamine release without addition of phosphatidylserine, and the in vivo administration of NGF to rats causes both connective tissue and mucosal mast cells to dramatically increase in number. All of these effects are both dose dependent and NGF specific, as evidenced by inhibition with anti-NGF. 2.5S NGF also causes in vitro increase of colonies in methylcellulose cultures of human peripheral blood. The effects of NGF in this system are synergistic with other T cell-derived growth factors and relatively specific for metachromatic cell growth. These observations support the conclusions that nerves and mast cells may constantly communicate and provide a structural and conceptual framework whereby the central nervous system may communicate with inflammatory events.

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.

Vagal Afferent Nerve Fibres Contact Mast Cells in Rat Small Intestinal Mucosa

Mast cells degranulate when exposed to specific antigens (via surface bound IgE), resulting in the release of numerous pro-inflammatory mediators. Neuroregulatory substances also activate mast cells, and may effect differential mediator release, without degranulation, suggesting a role for nerves in modulating mast cell activity. We previously investigated the microanatomical relationships of intestinal mucosal mast cells (IMMC) with nerves and found extensive associations in the intestinal mucosae of rats and humans. The origins of nerves that contact IMMC have not been determined; however, recent morphological and functional studies suggest the possibility that the vagus nerve might be involved. In the current study we show that vagal afferent fibers (labeled by injecting DiI into the nodose ganglion) penetrate to the tips of jejunal villi; and that some of these nerves make intimate contact with IMMC. These data provide the microanatomical basis for direct neural communication between the central nervous system (CNS) and mast cells in the gastrointestinal mucosa.

An immunohistochemical study of pleomorphic adenomas of the salivary gland:: Glial fibrillary acidic protein-like immunoreactivity identifies a major myoepithelial component

An immunohistochemical study of 34 pleomorphic adenomas of the major salivary glands demonstrated phenotypic differences among the various morphologic regions in these tumors. The phenotypes expressed were comparable to those of normal salivary gland cells. In the normal glands, myoepithelial cells were immunoreactive for glial fibrillary acidic protein (GFAP), S-100 protein, and keratin; acinic cells exhibited strong, predominantly nuclear S-100 staining and weaker keratin staining; intercalated ducts had both cytoplasmic and nuclear S-100 positivity; and several epithelial antigens were observed throughout the ductal system. In the tumors, the presence of classic epithelial markers (including carcinoembryonic antigen, epithelial membrane antigen, secretory component, and keratin) in the luminal cells of ducts and the intense immunoreactivity with GFAP (with weaker keratin and S-100 staining) in periductal and stromal cells indicated distinct epithelial and myoepithelial differentiation. Solid epithelioid areas consisted phenotypically of intercalated duct/acinic cells and/or myoepithelial cells, the former exhibiting predominant nuclear S-100 positivity. The presence of GFAP-like immunoreactivity in normal myoepithelial cells strongly supports the extensive involvement of this cell in pleomorphic adenomas. The spectrum of phenotypes expressed adds weight to existing evidence for pleomorphism rather than a mixed origin of this tumor. The combination of keratin, S-100, and GFAP immunostaining is particularly useful in identifying the component cells in pleomorphic adenomas of the salivary glands.

Nerve Remodelling during Intestinal Inflammationa

The intestinal mucosa contains a dense nerve network and many inflammatory cells, and these may interact through the exchange of regulatory molecules. Evidence suggests that intestinal mucosal mast cells are innervated, and it is known that the density of this cell type changes significantly in nematode-infected rats. Recent data indicates that rat jejunal mucosal nerves remodel after Nippostrongylus brasiliensis infection, with degenerative and regenerative phases during the acute and recovery stages of inflammation. Seven weeks postinfection there is a net increase in the density and number per villus of mucosal nerves. These changes suggest that mucosal nerves exhibit structural plasticity in inflamed tissues, which must impact on interactions between the enteric nervous system and other mucosal elements in disease.

Electrical stimulation of the vagus nerve modulates the histamine content of mast cells in the rat jejunal mucosa.

Mast cells are best known for their participation in allergic reactions. However, a number of recent studies suggest that mast cells are subject to nervous control. In the gut mucosa, mast cells are intimately associated with nerves, and the psychologically conditioned release of RMCP II (a mucosal mast cell-derived mediator) has been reported. These data suggest the potential for CNS regulation of intestinal mucosal mast cells. In this study, we stimulated the cervical vagi and found an increased histamine content in mucosal mast cells, without apparent degranulation. Furthermore, these changes could be prevented by subdiaphragmatic vagotomy. These data support the potential for intestinal mucosal mast cell regulation by the central nervous system and suggest modulation of mast cells without degranulation.

Effect of truncal vagotomy and capsaicin on mast cells and IgA-positive plasma cells in rat jejunal mucosa

Immunocompetent cells, including mast cells and plasma cells (PC), in the intestinal mucosa are closely apposed to nerve fibres. Recent work has shown that vagal afferent nerves penetrate the jejunal mucosa and contact intestinal mucosal mast cells (IMMC); and that electrical stimulation of the vagus results in increased IMMC histamine content. To determine if the vagus nerve exerts a trophic effect on immunocompetent cells in the gut mucosa, the effects of truncal vagotomy and neonatal capsaicin treatment on IMMC and IgA containing PC in the lamina propria of rat jejunum were investigated. Three weeks after vagotomy, microdensitometric assessment of Alcian blue stained sections revealed 25% fewer IMMC in vagotomized animals than in controls (P < 0.05). Three months after neonatal capsaicin administration 28% fewer IMMC were found in treated rat jejunum, compared with littermate controls (P < 0.05). Three weeks post‐surgery, IgA‐PC densities were increased in both vagotomized animals (that also underwent pyloroplasty) and pyloroplasty controls, compared to animals subjected to laparotomy only. The proportion of lamina propria areas remained stable, indicating that the observations reflected real reductions in the numbers of IMMC. We also determined the densities of B‐50 (a nerve growth‐associated protein, also called GAP‐43) immunoreactive nerve fibres in the lamina propria, as well as nerve profile areas, three weeks after vagotomy, and these parameters were unchanged. Taken together, these findings support the hypothesis that the vagus exerts a trophic effect on IMMC; and that capsaicin‐sensitive nerves also affect the IMMC population. These data add to the growing body of evidence for a functional connection between IMMC and the nervous system.

Nerve growth factor enhances antigen and other secretagogue-induced histamine release from rat peritoneal mast cells in the absence of phosphatidylserine.

The effects of 2.5S nerve growth factor (NGF) and epidermal growth factor (EGF), isolated from mouse submaxillary glands, on histamine release from rat peritoneal mast cells (PMCs) were studied. In the absence of phosphatidylserine, NGF (1 ng/ml to 1 microgram/ml) did not cause histamine release from PMCs isolated from normal rats and those infected with the nematode Nippostrongylus brasiliensis. However, when PMCs (greater than 97% pure) were preincubated with NGF and then challenged with worm antigen (Ag), there was a marked enhancement of histamine release (approximately twofold with a maximum effect at 10 ng/ml of NGF [3.8 X 10(-10) mol/L]) compared with the release induced by Ag alone. EGF (1 ng/ml to 1 microgram/ml) neither produced histamine release from PMCs in the presence of phosphatidylserine nor enhanced Ag-induced histamine release. This suggests that NGF acts directly on PMCs by activation of cell-surface receptors. The early kinetics of Ag-induced histamine release were altered by NGF that increased the initial rate at 15 seconds but did not prolong the overall duration of histamine release. Simultaneous addition of Ag and NGF did not cause enhanced histamine release; thus, some preincubation time with NGF (5 minutes or less) was required for the activation of PMCs. Moreover, after PMCs were activated by NGF, that state persisted for 1 hour, even when unbound NGF was removed by washing, and thereafter subsided gradually. Further studies revealed that NGF enhanced histamine release induced by concanavalin A, compound 48/80, and ionophore A23187. These results suggest that NGF might be an important molecule in inflammatory responses through the regulation of mediator release from mast cells.

Joint pathology and behavioral performance in autoimmune MRL-lpr mice

Young autoimmune MRL-lpr mice perform more poorly than age-matched controls in tests of exploration, spatial learning, and emotional reactivity. Impaired behavioral performance coincides temporally with hyperproduction of autoantibodies, infiltration of lymphoid cells into the brain, and mild arthritic-like changes in hind paws. Although CNS mechanisms have been suggested to mediate behavioral deficits, it was not clear whether mild joint pathology significantly affected behavioral performance. Previously we observed that 11-week-old MRL-lpr mice showed a trend for disturbed performance when crossing a narrow beam. The first aim of the present study was to test the significance of this trend by increasing the sample size and, second, to examine the possibility that arthritis-like changes interfere with performance in brief locomotor tasks. For the purpose of the second goal, 18-week-old mice that differ widely in severity of joint disease were selectively taken from the population and tested in beam walking and swimming tasks. It was expected that the severity of joint inflammation would be positively correlated with the degree of locomotor impairment. The larger sample size revealed that young MRL-lpr mice perform significantly more poorly than controls on the beam-walking test, as evidenced by more foot slips and longer traversing time. However, significant correlation between joint pathology scores and measures of locomotion could not be detected. The lack of such relationship suggests that mild joint pathology does not significantly contribute to impaired performance in young, autoimmune MRL-lpr mice tested in short behavioral tasks.