TLR2 Plays a Pivotal Role in Mediating Mucosal Serotonin Production in the Gut.

Serotonin (5-hydroxytryptamine [5-HT]) is a key enteric signaling molecule that mediates various physiological processes in the gut. Enterochromaffin (EC) cells in the mucosal layer of the gut are the main source of 5-HT in the body and are situated in close proximity to the gut microbiota. In this study, we identify a pivotal role of TLR2 in 5-HT production in the gut. Antibiotic treatment reduces EC cell numbers and 5-HT levels in naive C57BL/6 mice, which is associated with downregulation of TLR2 expression but not TLR1 or TLR4. TLR2-deficient (Tlr2 -/-) and Myd88 -/- mice express lower EC cell numbers and 5-HT levels, whereas treatment with TLR2/1 agonist upregulates 5-HT production in irradiated C57BL/6 mice, which are reconstituted with Tlr2 -/- bone marrow cells, and in germ-free mice. Human EC cell line (BON-1 cells) release higher 5-HT upon TLR2/1 agonist via NF-κB pathway. Tlr2 -/- mice and anti-TLR2 Ab-treated mice infected with enteric parasite, Trichuris muris, exhibited attenuated 5-HT production, compared with infected wild-type mice. Moreover, excretory-secretory products from T. muris induce higher 5-HT production in BON-1 cells via TLR2 in a dose-dependent manner, whereby the effect of excretory-secretory products is abrogated by TLR2 antagonist. These findings not only suggest an important role of TLR2 in mucosal 5-HT production in the gut by resident microbiota as well as by a nematode parasite but also provide, to our knowledge, novel information on the potential benefits of targeting TLR2 in various gut disorders that exhibit aberrant 5-HT signaling.

Modulation of the immune response by helminths: a role for serotonin?

The mammalian gut is a remarkable organ: with a nervous system that rivals the spinal cord, it is the body's largest repository of immune and endocrine cells and houses an immense and complex microbiota. Infection with helminth parasites elicits a conserved program of effector and regulatory immune responses to eradicate the worm, limit tissue damage, and return the gut to homeostasis. Discrete changes in the nervous system, and to a lesser extent the enteroendocrine system, occur following helminth infection but the importance of these adaptations in expelling the worm is poorly understood. Approximately 90% of the body's serotonin (5-hydroxytryptamine (5-HT)) is made in enterochromaffin (EC) cells in the gut, indicative of the importance of this amine in intestinal function. Signaling via a plethora of receptor subtypes, substantial evidence illustrates that 5-HT affects immunity. A small number of studies document changes in 5-HT levels following infection with helminth parasites, but these have not been complemented by an understanding of the role of 5-HT in the host-parasite interaction. In reviewing this area, the gap in knowledge of how changes in the enteric serotonergic system affects the outcome of infection with intestinal helminths is apparent. We present this as a call-to-action by investigators in the field. We contend that neuronal EC cell-immune interactions in the gut are essential in maintaining homeostasis and, when perturbed, contribute to pathophysiology. The full affect of infection with helminth parasites needs to define, and then mechanistically dissect the role of the enteric nervous and enteroendocrine systems of the gut.

Immune response in irritable bowel syndrome: A systematic review of systemic and mucosal inflammatory mediators.

OBJECTIVE: To systematically review the available data on cytokine and immune cells in the peripheral blood and mucosal biopsy samples from patients with irritable bowel syndrome (IBS). METHODS: From a review of the literature, data on cytokines and immune cells that had been assayed in at least three independent studies were collated and trends examined. RESULTS: Levels of interleukin (IL)-10 tended to be decreased and those of IL-6, IL-8, tumor necrosis factor-α and IL-1ß increased in the systemic circulation in IBS, while in the mucosa, IL-10 was decreased and IL-8, mast cells, enterochromaffin cells and CD3+ T lymphocytes were increased. However, these findings were not consistent across all studies and, in some instances, were limited to certain IBS sub-populations. CONCLUSIONS: The interpretation of this literature is limited by several factors, such as the intrinsic heterogeneity of IBS and a lack of standardization in study design. While a number of intriguing immunological observations have been made in IBS, more work is needed before a compelling case can be made for a role for immune-mediated events in the etiology of IBS.

The role of serotonin and its receptors in activation of immune responses and inflammation.

Serotonin or 5-hydroxytryptamine (5-HT) is a neurotransmitter and hormone that contributes to the regulation of various physiological functions by its actions in the central nervous system (CNS) and in the respective organ systems. Peripheral 5-HT is predominantly produced by enterochromaffin (EC) cells of the gastrointestinal (GI) tract. These gut-resident cells produce much more 5-HT than all neuronal and other sources combined, establishing EC cells as the main source of this biogenic amine in the human body. Peripheral 5-HT is also a potent immune modulator and affects various immune cells through its receptors and via the recently identified process of serotonylation. Alterations in 5-HT signalling have been described in inflammatory conditions of the gut, such as inflammatory bowel disease. The association between 5-HT and inflammation, however, is not limited to the gut, as changes in 5-HT levels have also been reported in patients with allergic airway inflammation and rheumatoid arthritis. Based on searches for terms such as '5-HT', 'EC cell', 'immune cells' and 'inflammation' in pubmed.gov as well as by utilizing pertinent reviews, the current review aims to provide an update on the role of 5-HT in biological functions with a particular focus on immune activation and inflammation.

Anticolitis activity of Chinese herbal formula yupingfeng powder via regulating colonic enterochromaffin cells and serotonin.

OBJECTIVE: To investigate whether traditional Chinese herbal formula Yupingfeng (YPF) powder has an anti-inflammatory effect on colonic inflammation, and to explore the mechanism involved. MATERIALS AND METHODS: YPF powder was orally administrated to trinitrobenzene sulfonic acid (TNBS)-induced colitis mice at the dose of 3, 6, and 12 g/kg/d for 7 consecutive days. Body weight, stool consistency, histopathological score, and myeloperoxidase (MPO) activity were tested to evaluate the effect of YPF powder on colonic inflammation while colonic enterochromaffin (EC) cell density and serotonin 5-hydroxytryptamine (5-HT) content were investigated to identify the effect of YPF powder on colonic 5-HT availability. RESULTS: The results showed that the body weight of colitis mice was markedly decreased by 10, 12, 14, and 17% at 1, 3, 5, and 7 days (P < 0.05), whereas stool consistency score (3.6 vs. 0.4, P < 0.05), histopathological score (3.6 vs. 0.3, P < 0.05), and MPO activity (2.7 vs. 0.1, P < 0.05) in colitis mice were significantly increased compared to that of the normal mice; YPF powder treatment dose-dependently increased the body weight (7-13% increase) and decreased the stool consistency score (0.4-1.4 decrease), histopathological score (0.2-0.7 decrease), and MPO activity (0.1-0.9 decrease) in colitis mice. Colonic EC cell density (70% increase) and 5-HT content (40% increase) were markedly increased in colitis mice (P < 0.05), YPF powder treatment dose-dependently reduced EC cell density (20-50% decrease), and 5-HT content (5-27% decrease) in colitis mice. CONCLUSION: The findings demonstrate that the anti-inflammatory effect of YPF powder on TNBS - induced colitis may be mediated via reducing EC cell hyperplasia and 5-HT content. The important role of YPF powder in regulating colonic EC cell number and 5-HT content may provide an alternative therapy for colonic inflammation.

[Serotonin and its immune and physiological effects].

Now that the neurotransmitter serotonin modulates the immune system cells, and its main sources for antigenpresenting cells and lymphocytes are enterochromaffin cells of the gut, peripheral nerves, platelets and mast cells in case of inflammation. Immune cells uptake serotonin because they express receptors for this monoamine and intracellular serotonin transporters. The dendritic cells have a mechanism to transfer serotonin to T lymphocytes during antigen presentation. The macrophages and T cells have the ability to serotonin synthesis. Serotonin can influence mobility and proliferation of lymphocytes, phagocytosis, cytolytic properties, synthesis of chemokines and cytokines. Diversity of immunomodulating effects of serotonin is determined by heterogeneity of serotoninergic receptors. Immunomodulating action of serotonin is evidence of the close relationship between nervous and immune systems.

Tryptophan hydroxylase autoantibodies as markers of a distinct autoimmune gastrointestinal component of autoimmune polyendocrine syndrome type 1.

BACKGROUND: Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract. AIMS: We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID. METHODS: TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy. RESULTS: Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels. CONCLUSIONS: TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

ZBP-89 regulates expression of tryptophan hydroxylase I and mucosal defense against Salmonella typhimurium in mice.

BACKGROUND & AIMS: ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium. METHODS: We generated mice with a conditional null allele of Zfp148 (ZBP-89(FL/FL)) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89(FL/FL)), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89(ΔInt) and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S. typhimurium after administration of streptomycin. RESULTS: Microarray analysis revealed that the colonic mucosa of ZBP-89(ΔInt) mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [5HT]) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89(ΔInt) mice did not increase mucosal levels of 5HT in response to S. typhimurium infection, and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S. typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89(ΔInt), mice, correlating with fecal levels of butyrate. CONCLUSIONS: ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis after infection with S. typhimurium, partly because of reduced induction of 5HT production in response to butyrate and decreased secretion of antimicrobial peptides.

IL-13-mediated immunological control of enterochromaffin cell hyperplasia and serotonin production in the gut.

Enterochromaffin (EC) cells in the gastrointestinal (GI) mucosa are the main source of serotonin (5-hydroxytryptamine (5-HT)) in the body. 5-HT is implicated in the pathophysiology of many GI disorders including functional and inflammatory bowel disorders. Herein we studied the role of interleukin 13 (IL-13) in EC cell biology by utilizing IL-13-deficient (IL-13-/-) mice and BON cells (a model for human EC cells). The numbers of EC cells and 5-HT amount were significantly lower in enteric parasite, Trichuris muris-infected IL-13-/- mice compared with the wild-type mice. This was accompanied with increased parasite burden in IL-13-/- mice. Treatment of naive and infected IL-13-/- mice with IL-13 increased EC cell numbers and 5-HT amount. BON cells expressed IL-13 receptor and in response to IL-13 produced more 5-HT. These results provide novel information on IL-13-mediated immunological control of 5-HT in the gut, which may ultimately lead to improved therapeutic opportunities in various GI disorders.

Enterochromaffin cell hyperplasia in irritable pouch syndrome.

BACKGROUND: Irritable pouch syndrome (IPS) is a functional disease in patients with ileal pouch-anal anastomosis following colectomy for ulcerative colitis (UC). The pathophysiology of IPS is characterized by the presence of visceral hypersensitivity, similar to that seen in irritable bowel syndrome. However, the exact etiology and pathogenesis of IPS are not known. We hypothesized that serotonin-containing enteroendocrine cells or enterochromaffin (EC) cell hyperplasia and alterations in the mucosal immune cells may contribute to the patients' symptoms. The aim of the study was to assess EC cell hyperplasia and alterations in the mucosal immune cells in IPS. METHODS: The Pouchitis Disease Activity Index (PDAI) was used to quantify symptoms and mucosal inflammation in 36 patients with IPS and 25 patients with normal pouches. The histology and immunohistochemistry of pouch mucosal biopsies were assessed by a blinded gastrointestinal pathologist for intraepithelial lymphocytes (IEL), CD3+ T cells, CD25- (interleukin [IL]-2 receptor), tryptase- (mast cells), and serotonin-expressing cells. The numbers of IEL and immune-stained cells were compared between the two groups. RESULTS: Both groups were compatible demographically in terms of age, gender, duration of UC, stage, indication, and duration of the pouch surgery. There were no differences in the number of IEL, CD3+ T cells, CD25+ cells, and mast cells between the IPS and normal control groups. However, there were a significantly larger number of EC cells in the IPS group than that in the control group (54.8 +/- 24.9 vs 36.7 +/- 17.5 per 4 200x epithelial cells, P < 0.005). The number of EC cells appeared to be correlated with the symptom score (r = 0.276, P= 0.032). There were no significant correlations between the PDAI endoscopy and histology scores and the number of EC cells or between the PDAI scores and the number of IEL or other immune-stained cells. CONCLUSIONS: A greater number of EC cells were found in the IPS group than the normal pouch group, and the number of EC cells appeared to be correlated with the clinical symptoms of IPS. EC cell hyperplasia may be a contributing mechanism of visceral hypersensitivity and symptoms in IPS.

Proliferative capacity of enterochromaffin cells in guinea-pigs with experimental ileitis.

Enterochromaffin (EC) cells regulate gut motility and secretion in response to luminal stimuli, via the release of serotonin (5-HT). Inflammatory bowel disease and other gastrointestinal disorders are associated with increased numbers of EC cells and 5-HT availability. Our aim was to determine whether proliferation of EC cells contributed to the hyperplasia associated with intestinal inflammation. Ileitis was induced in guinea-pigs by intraluminal injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS). A single pulse of 5-bromo-2'-deoxyuridine (BrdU) was injected 1 or 24 h before the collection of tissue, 6 or 7 days after TNBS treatment. In the controls, the labelling index (percentage of BrdU-labelled EC cells) was less than 1%. Despite a significant increase in EC cells in the inflamed ileum, the labelling index was similar in the TNBS-treated animals to that of controls. An increased occurrence of EC cells in the BrdU-labelled zone accounted for the increase in EC cells in the inflamed ileum. Goblet cell numbers were also significantly increased in the inflamed ileum, indicating that cell hyperplasia was not limited to the enteroendocrine cell lineage. This study demonstrates that a small portion of EC cells retain some proliferative capacity; however, hyperplasia associated with ileitis is not attributable to the increased proliferation of EC cells and is not limited to one cell lineage. Therefore, EC cell hyperplasia most probably occurs at the level of the stem cell or recruitment from the progenitor pool.

CD4+ T cell-mediated immunological control of enterochromaffin cell hyperplasia and 5-hydroxytryptamine production in enteric infection.

BACKGROUND: Enterochromaffin (EC) cells are dispersed throughout the gastrointestinal (GI) mucosa and are the main source of 5-hydroxytryptamine (5-HT) in the gut. 5-HT has been implicated in the pathophysiology of several GI disorders, but the mechanisms regulating 5-HT production in the gut are unknown. AIM: To investigate the role of CD4(+) T cells in the production of 5-HT using a model of enteric parasitic infection. METHODS AND RESULTS: Severe combined immunodeficient (SCID) mice and their wild-type controls were infected with the nematode Trichuris muris and killed on various days after infection to study colonic EC cells and 5-HT production. The number of EC cells and the amount of 5-HT produced were significantly higher in infected wild-type mice than in non-infected mice. The number of EC cells and the amount of 5-HT after infection were significantly lower in SCID mice after infection than in wild-type mice. The number of EC cells and the amount of 5-HT was significantly increased after reconstitution of SCID mice with CD4(+) T cells from infected mice and this was accompanied by an upregulation of colonic CD3 T cells and T helper 2 (Th2) cytokines. Laser capture microdissection-based molecular and immunofluorescence techniques revealed the presence of interleukin 13 receptor alpha1-chain on EC cells. CONCLUSION: These results show an important immunoendocrine axis in the gut, where secretory products from CD4(+) T cells interact with EC cells to enhance the production of 5-HT in the gut via Th2-based mechanisms. These results show new insights into the mechanisms of gut function, which may ultimately lead to improved therapeutic strategies in functional and inflammatory disorders of the GI tract.

[Influence of melatonin, serotonin and gastrin on streaming of chronic gastritis at elderly patients].

In development of chronic gastritis of elderly persons there has been increased the functional activity of diffuse neuroendocrine systems, especially EC-cells. It results in decreasing of the indexes of proliferationand increasing of apoptosis index. At patients with chronic gastritis in elderly, we found diffuse lesion of gastric mucous, combination with intestinal methaplasy and atrophy of stomach mucous more often. The further studying of processes of cellular updating and mechanisms of regulation of apoptosis and proliferation will allow stopping the development of chronic gastritis and occurrence of its irreversible complications at early stage.

Close encounters of the monoamine kind: immune cells betray their nervous disposition.

Here we review the evidence for immune cells expressing multiple components of the serotonergic and dopaminergic systems that are more commonly associated with the central nervous system (CNS). We discuss where and how peripheral encounters with these biogenic monoamines occur and posit reasons as to why the immune system would wish to deploy these pathways. A full taxonomy of serotonergic and dopaminergic constituents and their workings in component cells of the immune system should facilitate the formulation of novel therapeutic approaches in diseases characterized by immune dysfunction and potentially provide a range of surrogate peripheral markers for registering and monitoring disturbances within the CNS.

Proximity between 5-HT secreting enteroendocrine cells and lymphocytes in the gut mucosa of rhesus macaques (Macaca mulatta) is suggestive of a role for enterochromaffin cell 5-HT in mucosal immunity.

The involvement of serotonin (5-hydroxytryptamine; 5-HT) in immunoregulation has been well documented. Gut mucosa is a large reservoir of 5-HT most of which is attributed to gut endocrine cells. In this study, we examined the anatomical relationship among 5-HT immunoreactive cells and T and B lymphocytes in the gut mucosa of rhesus monkeys (Macaca mulatta). 5-HT, CD3 and CD20 immunoreactive cells were immunofluorescently labeled and visualized by confocal microscopy. 5-HT immunoreactive cells were primarily found within the epithelium of the intestine and were present at all levels of the gastrointestinal tract. Many 5-HT immunoreactive cells were in contact with, or very close proximity to CD3(+) and CD20(+) lymphocytes. These results provide morphological evidence to suggest interactions between 5-HT secreting enteroendocrine cells and lymphocytes in the gut mucosa. This further supports a possible role of 5-HT in mucosal immune responses.

Transmembrane protein tyrosine phosphatase IA-2 (ICA512) is expressed in human midgut carcinoids but is not detectable in normal enterochromaffin cells.

A potential upregulation of receptor type protein tyrosine phosphatase IA-2 (ICA512) expression was detected by differential display and investigated in midgut carcinoid tumours. Normal intestine tissue and tumour tissue from 13 midgut carcinoid patients were studied by in situ hybridisation using an IA-2 ribonucleotide probe and confocal microscopy using specific IA-2 antibodies. Previously, it had been shown that IA-2 is located in the secretory granules of virtually all neuroendocrine cells. However, we found that IA-2 was not detectable in resting normal enterochromaffin (EC) cells of the small intestine, while high expression of IA-2 mRNA and protein was confirmed in both primary and metastatic carcinoid tissue. This difference in expression was not observed with chromogranin A or serotonin, two secretory granule hormones known to be expressed in EC cells, indicating that IA-2 was seemingly not necessary for the basal production and packaging of these hormones. When comparing patients receiving biotherapy before operation with untreated patients, we found expression of IA-2 to be lower in tumours from patients that had been treated with a combination of alpha-interferon and the somatostatin analogue, octreotide. There was no correlation between IA-2 expression and proliferation rates as measured by immunohistochemistry with antibodies against the Ki 67 antigen. Furthermore, we show that IA-2 is co-localised with serotonin in carcinoid tumours as well as in the pancreatic tumour cell line, BON1, which is interesting as serotonin secretion rate is presumably higher in tumour cells than in resting EC cells. Taken together, these findings may indicate a role for IA-2 in the later stages of the regulated secretory process.

Immunocytochemical distribution of serotonin and neuropeptide Y (NPY) in mouse adrenal gland.

By the use of immunocytochemical staining methods, we studied the morphology and distribution of 5HT and NPY immunoreactive cells and fibres in the mouse adrenal gland. The 5HT-immunoreactive cells were numerous and widely localized in the medullar tissue. These cells were arranged in three cellular types with regard to their morphological and immunocytochemical features. One of them showed cells with polygonal shape, being intensified like the typical medullary chromaffin cells. These immunoreactive cells were observed arranged in medullar islets. The second 5HT-immunoreactive cellular type was constituted by cells with polygonal shape and strong immunoreactivity. The third one was formed by cells with immunoreactive prolongations. We found some islets of chromaffin non-immunoreactive cells surrounded by immunostained cells. We also observed some 5HT-immunoreactive nerve fibres in the medullar tissue. NPY-like immunoreactivity was detected in both chromaffin and ganglion cells in adrenal medulla. NPY-like immunoreactivity was also detected in nerve fibres at cortical level. In a few cases, we observed medullar 5HT- and NPY-immunoreactive tissue in the adrenal cortex (monotremas).

[Cellular and humoral immunity in duodenal peptic ulcer with gastrin cell hyperplasia]. / Kletochnyi i gumoral'nyi immunitet pri iazvennoi bolezni dvenadtsatiperstnoi kishki s giperplaziei gastrinovykh kletok.

A total of 61 patients with frequently relapsing duodenal ulcer were examined. Of these, 18 patients were in the acute phase and 43 experienced remission. Using 1 mm2 of the mucosa, measurements were made of the counts of duodenal and pyloric G-cells (by immunomorphologic assay), of the absolute and relative counts of T and B lymphocytes, the content of IgA, IgM and IgG, histamine and serotonin (by fluorometry) in the blood, and of the concentration of uropepsin in the urine. In the stages of exacerbation and remission, the patients suffering from duodenal ulcer with hyperplasia of G-cells manifested, as compared with the analogous patients without hyperplasia, a decrease of the absolute and relative counts of T cells, especially of those of B cells, combined with a rise of the content of IgM and IgG during exacerbation, followed by its returning to normal in the phase of remission. Over one year part of the duodenal ulcer patients with hyperplasia of G-cells received preventive treatment with ranitidine, which resulted in a tendency towards the lowering of the count of pyloric G-cells and the rise of the absolute and relative counts of T cells.

Monoclonal antibodies to chromaffin cells can distinguish proteins specific to or specifically excluded from chromaffin granules.

I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.

Amines of the mucosal mast cell of the gut in normal and nematode infected rats.

Infection with the nematode N. brasiliensis is accompanied by a marked increase of the number of mucosal mast cells (MMC) and the mucosal content of histamine and 5-hydroxytryptamine (5-HT). We compared amine levels, determined by ion exchange and high performance liquid chromatography (HPLC) with numbers of MMC and enterochromaffin cells (ECC). Furthermore, we measured 5-HT cytofluorometrically in individual MMC and ECC. The cellular distribution of 5-HT was studied immunohistochemically. Our results corroborate previous findings that histamine is stored in MMC. Quotients between histamine content and numbers of MMC decreased throughout the period of worm expulsion, followed by a recovery, suggesting a histamine release during this defense reaction. The HPLC analysis gave no evidence for a storage of dopamine in MMC. ECC and MMC of normal and infected rats showed a formaldehyde induced fluorescence and 5-HT immunoreactivity. The formaldehyde induced fluorescence of MMC from normal rats was about 10% that of ECC, but MMC exceeded ECC three times by numbers. These findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC. ECC numbers did not change during the infection and their content of 5-HT was unchanged, as judged by cytofluorometry. The cytofluorometric measurements showed that the intensity of the monoamine fluorescence from the MMC of infected animals was about three times as high as that of controls. It was concluded that the increased tissue levels of 5-HT was due to both an increase in MMC numbers and an increase in the 5-HT content of individual MMC. The results suggest a different role for histamine and 5-HT in the defense reaction towards the nematode infection.