Association between histamine-containing mast cells and sensory nerves in the skin and airways of control and capsaicin-treated pigs

Summary The association between mast cells (visualized by routine staining and immunohistochemistry for histamine) and capsaicin-sensitive nerves (containing calcitonin gene-related peptide (CGRP) and substance P (SP)) was studied in the pig. In the 1-ethyl-3(3-diethylaminopropyl)carbodiimide (EDCDI)-fixed skin tissue, histamine-containing mast cells and CGRP/SP-positive nerves were found in close association around blood vessels. In the EDCDI-fixed airway mucosa, only single histamine-containing mast cells were detected. However, many alcian blue-positive mast cells were found, sometimes close to the airway epithelium where CGRP/SP-containing nerve fibres were absent 2 days after systemic capsaicin pretreatment, but no changes in the number and distribution of tissue mast cells, granulocytes or lymphocytes, or the number of blood leukocytes were detected. Local injection of allergen, histamine and capsaicin into the skin of pigs actively sensitized with ascaris antigen caused a rapid light red-flare (vasodilation) reaction. Allergen and histamine, but not capsaicin, also produced plasma protein extravasation. In contrast to the absent flare, the protein extravasation response still occurred in capsaicin-treated pigs. The sensitivity to ascaris antigen was mediated by an IgE-like antibody. We conclude that a functional and morphological relationship exists between histamine-containing mast cells and capsaicin-sensitive sensory nerves in the pig skin. Mast cells and sensory nerves are also found in the airway mucosa and appear to be closely associated with the epithelium.     

Endoneurial mast cells in peripheral nerves of the armadillo dermis

Summary Electron microscopy of the dermis of the 9-banded armadillo, Dasypus novemcinctus, reveals an intimate relationship of mast cells to peripheral nerves. Mast cells are routinely found in the dermis in close proximity to nerves, and mast cells are also present within the perineurial cell sheath in the endoneurial space proper. These cells contain electron opaque granules and exhibit numerous surface folds as is characteristic of other species. The degranulation of the mast cells by injection of 0.5% aqueous trypan blue reveals sequential exocytosis of granules, similar to that described for the rat. Degranulation occurs in most dermal and endoneurial mast cells following a single intradermal injection of trypan blue. A small number of mast cells partially degranulate and a few exhibit no degranulation, suggesting a heterogeneous population. It is also noted that mitochondrial morphology differs in degranulating cells, in that they become swollen and vacuolated. The results of this study are discussed in light of previous results reported for other species, mainly at the light microscope level.Supported by institutional grant from the L.S.U. School of DentistryThe author thanks Ivis Insua and Angela Pepin for their technical and secretarial assistance and Garbis Kerimian for his photographic work     

Small intensely fluorescent (SIF) cells and sympathetic nerves in the adult rabbit portal vein and during perinatal development

Summary The ontogenesis of small intensely fluorescent (SIF) cells and the adrenergic nerve plexus is described in stretch preparations of the rabbit portal vein. On the 25 to 26th days of gestation there was a predominance of SIF cells (8 to 30 m in diameter), but a few nerve fibres in bundles were also present. Each portal vein preparation contained 6 to 9 groups of cells. The distribution and number of SIF cells and nerve bundles remained constant until the 31st day of gestation at which stage the number of SIF cells had decreased, while the density of the nerve plexus had increased approximately 4-fold. The adult portal vein exhibited a dense adrenergic plexus, but SIF cells were absent from nine out of ten preparations.     

Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells

Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer’s patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2±3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.This work was supported by the EU (5th framework programme “Healthypigut” QLK5-CT-2000-00522 and 6th framework programme “Feed for Pig Health” FOOD-CT 2004-506144).     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

Frequency of mast cells in the olfactory mucosa of rhesus monkeys

During studies of the olfactory mucosa and its response to the different levels of circulating sex hormones, considerable numbers of mast cells have been observed in its epithelia and subepithelial regions. The number of these cells in the olfactory mucosa of male monkeys differs greatly from that found in females. The frequency of these cells in the olfactory mucosa of females fluctuates significantly during the menstrual cycle. These fluctations stimultaneously correspond to the well known changes in olfactory sensitivity: around ovulation, when the olfactory sensitivity for certain odorants is high, the number of mast cells in the olfactory mucosa also increases.     

Synthesis and pharmacological evaluation of the individual stereoisomers of 3-[methyl(1,2,3,4-tetrahydro-2-naphthalenyl)amino]-1-indanone, a potent mast cell stabilising agent

Each stereoisomer of 3-[methyl(1,2,3,4-tetrahydro-2-naphthalenyl)amino]-1-indanone, 1a-d, was prepared and evaluated in vitro for its ability to prevent mediator release induced by different degranulating agents from rodent mast cells and also in vivo against passive cutaneous anaphylaxis. The manner in which the stereoisomers prevented direct membrane activation was found to be highly dependent on the stereochemistry of the individual isomers. Stereoisomer 1b was the most active isomer in vivo, exhibiting superior activity to disodium cromoglycate.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

Electron microscopic study of the regeneration in vitro of rat peritoneal mast cells after histamine secretion

Summary Regeneration of rat mast cells was studied by TEM from 10 s to 48 h after secretion of histamine induced by compound 48/80. During the first 2 h, small intracellular cavities, formed during compound exocytosis and containing non-membrane-bound remnants of the granules, tended to coalesce, and after 2 h of incubation regeneration started. After 6 h, all the cavities had fused into one large central cavity which contained the remnants of the granules and remained open to the exterior during the entire period. The plasma membrane microfolds which disappeared just after secretion were reformed during regeneration. They were apparently involved in endocytotic-like activity and coated vesicles also appeared beneath the plasmalemma (membrane recycling?). The fate of the granule remnants in the cavity is unknown, as regeneration was not completed after 48 h which is the longest survival time obtained so far in ultrastructural studies of mast cell regeneration in vitro.     

A quantitative cytochemical study of the growth of individual mast cells

Summary For individual mast cells, relationships between their dry mass and their content of heparin and 5-hydroxytryptamine (5-HT) were studied. This was achieved by measuring these parameters successively on identical cells, by means of quantitative cytochemical techniques. The peritoneal mast cells have a very long life span and a slow turnover of granule components. Increase of the dry weight of the cells may therefore be taken as an expression of cellular growth. Mast cell populations from younger and older animals were analysed in an attempt to evaluate the influence of cell-aging and animal-aging on the growth of the mast cells. The analysis was based on allometric (log-log) plots and linear regressions. Within the cell populations there were strong mutual correlations between the cell parameters studied, without any obvious deviations from linearity. However, the slopes of the allometric lines indicated a somewhat different mode of growth for mast cells from younger and older animals. The capacity of the mast cells to accumulate 5-HT after a single injection of its precursor, 5-hydroxytryptophan, was used as a functional test. In relation to the cell weight, the induced increase of 5-HT was greater for lighter than for heavier mast cells. This difference between light and heavy mast cells was greater for cells from younger than from older animals. These differences in growth and functional properties between mast cells from younger and older animals were interpreted as an effect of aging of the animals rather than of aging of the cells.Supported by grants from the Swedish Medical Research Council, Project no 2235I wish to express my sincere thanks to Professor Lennart Enerbäck for valuable and constructive criticism of this paper. I also wish to thank Lecturer Erik Leander for valuable statistical advice     

Morphological and functional characteristics of peritoneal mast cells from young rats

To study why neonatal and young rats are resistant to the effects of some secretagogues, such as compound 48/80 and 2.5-S nerve growth factor, we examined peritoneal mast cells from 14–15-day-old rats (young rats) and compared them to peritoneal mast cells from adults. Peritoneal mast cells from young rats contain approximately one-tenth of the amount of histamine observed in adult peritoneal mast cells. However, both cell populations contained similar low levels of the mucosal mast cell-associated protease rat mast cell protease II. Histochemical analysis of peritoneal mast cells from young rats using safranin O and berberine sulphate suggested that only a portion of the granules of these cells contained heparin. At an ultrastructural level the young rat peritoneal mast cell contains relatively few granules. The majority of mast cells from young rats have a bilobed or indented nucleus which is only rarely observed in adult cells. Functionally, the young rat peritoneal mast cell demonstrates a significantly reduced histamine release in response to the connective tissue mast cellspecific secretagogues compound 48/80 and 2.5-S nerve growth factor. In contrast, the percent histamine release in response to the neurotransmitter substance P, which degranulates both connective tissue mast cells and intestinal mucosal mast cells, was similar in the adult cells and the young rat cells. This study demonstrates substantial differences between the young rat and adult peritoneal mast cells which may explain the ability of very young animals to withstand large doses of certain secretagogues.     

Central projections of fibers in the auditory and tensor nerves of cicadas (Homoptera: Cicadidae)

Summary The auditory and tensor nerves of cicadas are mixed nerves containing both afferent and efferent elements. In 17-year cicadas, and in Okanagana rimosa, the auditory nerve contains afferents from body hairs, from the detensor tympani-chordotonal organ, and some 1300–1500 afferents from the hearing organ. Within the fused metathoracic-abdominal ganglionic complex the receptors from both the auditory and tensor nerves form a neuropilar structure that reveals the metameric organization of this complex. A few fibers run anteriorly, projecting into the meso and prothoracic ganglia. Within the ganglionic complex a division of auditory nerve afferents into a dense intermediate and a more diffuse ventral neuropile is observed. In addition, a dorsal motor neuropile is outlined by arborizations of the timbal motor neuron. This neuron is one of several efferent cell types associated with the auditory nerve, and there is an indication that several efferent fibers innervate the timbal muscle. There is anatomical evidence for a possible neuronal coupling between the bilaterally symmetrical large timbal motor neurons. In general, central projections from the auditory and tensor nerves support evidence of a structural layering within the CNS of insects.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride

Many batches of alcian blue dye are incompletely soluble at the low pH used for demonstrating mast cells. An improved technique using alcian blue tetrakis (methylpyridium) chloride (alcian blue pyridine variant) is described here. It produces stronger mast cell staining than other alcian blue stains tested.     

Synergistic augmentation of inflammatory cytokine productions from murine mast cells by monomeric IgE and toll-like receptor ligands

Simultaneous activation of murine mast cells by monomeric IgE and toll-like receptor (TLR) ligands was examined. Inflammatory cytokine production elicited by the binding of IgE in the absence of antigen, was further enhanced by the addition of lipopolysaccharide (LPS) or peptidoglycan (PGN). Enhancement by LPS or PGN on cytokine production was mediated by TLR4 and TLR2, respectively, since TLR4- and TLR2-deficient mast cells did not show synergistic activation by monomeric IgE and LPS/PGN. Synergistic activation of mast cells was obtained via phosphorylation of several mitogen-activated protein kinases (MAPK). Furthermore, MAPK inhibitors, significantly attenuated the augmentation of inflammatory cytokine production by monomeric IgE and LPS or PGN. Altogether, these results suggest that simultaneous TLR activation of mast cells with IgE molecules, particularly highly cytokinergic (HC) IgE, might contribute to the exacerbation of allergic diseases associated with infection even in the absence of a specific antigen.     

The mast cells of the mammalian central nervous system

Summary The uptake and turnover of the precursors of heparin and heparan sulphate (³⁵S), and of serotonin (³H-5-hydroxytryptophan; ³H-5-HTP) by mast cells (MCs) and neurolipomastocytoid cells (NLMs) of the mammalian CNS were studied. Rats of varying age from 1 day to early adulthood were injected with ³⁵S (as a solution of sodium sulphate) and ³H-5-HTP, and allowed to survive for different periods. Several fixatives, as well as lengths of exposure to photographic emulsion, were tested. The monoamine oxidase inhibitor, nialamide, needed to be given before uptake of ³H-5-HTP could be adequately demonstrated especially in the CNS. ³⁵S was taken up by structures known to contain a great deal of sulphate, viz., cartilage and goblet cells, as well as by MCs of adult liver and thymus, but not by MCs of adult CNS. All of these structures, including the MCs of CNS, took it up much more avidly in babies than in adults. ³H-5-HTP had a similar effect in that the MCs of younger animals took it up more strongly than did those of adults. In the MCs of the CNS uptake seemed to increase up to 15 days of age but then to decrease as maturity was reached. The MCs are located in the leptomeninges of the cerebral hemispheres as well as the choroid fissures and dorsal thalamus. The NLMs, ubiquitously distributed in the leptomeninges as well as perivascularly, showed less radioactivity with both markers in fewer cells and only in babies. The possible significance of these results is discussed. It is concluded that MCs, and to a lesser extent NLMs, of the CNS do permit entry of these markers, and that the more immature the cells, the heavier the load that enters. Adult cells do not seem to take up precursor suggesting little or no turnover.Supported in part by a grant from the Incentive Plan of the Medical School, American University of Beirut, and by Research Support Grant MA-004 from the College of Graduate Studies, University of Kuwait     

Differential usage of COX-1 and COX-2 in prostaglandin production by mast cells and basophils

Basophils have been erroneously considered as minor relatives of mast cells, due to some phenotypic similarity between them. While recent studies have revealed non-redundant roles for basophils in various immune responses, basophil-derived effector molecules, including lipid mediators, remain poorly characterized, compared to mast cell-derived ones. Here we analyzed and compared eicosanoids produced by mouse basophils and mast cells when stimulated with IgE plus allergens. The production of 5-LOX metabolites such as LTB4 and 5-HETE was detected as early as 0.5 h post-stimulation in both cell types, even though their amounts were much smaller in basophils than in mast cells. In contrast, basophils and mast cells showed distinct time course in the production of COX metabolites, including PGD2, PGE2 and 11-HETE. Their production by mast cells was detected at both 0.5 and 6 h post-stimulation while that by basophils was detectable only at 6 h. Of note, mast cells showed 8–9 times higher levels of COX-1 than did basophils at the resting status. In contrast to unaltered COX-1 expression with or without stimulation, COX-2 expression was up-regulated in both cell types upon activation. Importantly, when activated, basophils expressed 4–5 times higher levels of COX-2 than did mast cells. In accordance with these findings, the late-phase production of the COX metabolites by basophils was completely ablated by COX-2 inhibitor whereas the early-phase production by mast cells was blocked by COX-1 but not COX-2 inhibitor. Thus, the production of COX metabolites is differentially regulated by COX-1 and COX-2 in basophils and mast cells.