Michael G. Blennerhassett

Formation of contacts between mast cells and sympathetic neurons in vitro

SummaryFunctional interactions between mast cells and peripheral nerves may occur at sites of association seen in vivo. To study the interactions, we developed a tissue culture model of murine sympathetic neurons co-cultured with rat basophilic leukaemia (RBL-2H3) cells (homologues of mucosal mast cells) or rat peritoneal mast cells. In co-cultures of up to 3 days, light microscopy identified neurite contacts with peritoneal mast cells or RBL-2H3 cells, but not with glial cells or fibroblasts. Electron microscopy confirmed membrane-membrane contact between neurites and RBL-2H3 cells. Time-lapse analysis of interactions between neurons and RBL-2H3 cells showed that 60–100% of the cells in a given field acquired neurite contact within 17 h. In matching control studies, there was no increase in the frequency of neurite contact with cells of the rat plasmacytoma line (YB2/0): these were not selected as targets, and contacts were broken if formed. Time-lapse records of the derivation of neurites from their path suggested a neurotropic effect of mast cells, with neurite contact ensuing when the intervening distance was less than 36±4 μm. Once formed, contacts were invariably maintained throughout the period of examination (up to 72 h), in contrast to YB2/0 or fibroblast contacts. We conclude that neurons selectively form and maintain connections with cells representative of rat connective tissue-type and mucosal mast cells in vitro. Similar interactions in vivo could promote nerve/mast cell contacts, which may allow bidirectional communication between the nervous and immune systems.

Interleukin 1β induces the expression of interleukin 6 in rat intestinal smooth muscle cells

Abstract Background/Aims: The increased expression of several cytokines, including interleukin 6 (IL-6), has recently been reported in a study of the longitudinal muscle and myenteric plexus layers of rat intestine following Trichinella spiralis infection. However, the putative cellular sources and the mechanism underlying the induction of IL-6 in these tissues are presently unknown. The aim of this study was to examine the ability of cultured smooth muscle cells from rat jejunum to produce IL-6 messenger RNA and protein and to investigate the underlying mechanism. Methods: Cultured smooth muscle cells were treated with human recombinant interleukin 1β (HrIL-1β). The level of IL-6 messenger RNA was estimated by polymerase chain reaction, and the released IL-6 protein was estimated by bioassay. Results: HrIL-1β induced IL-6 messenger RNA expression in the smooth muscle cells in a time- and concentration-dependent manner. This was accompanied by the secretion of IL-6 protein into the medium. The effect of HrIL-1β was blocked by the IL-1 receptor antagonist, by actinomycin D, or by prior boiling of the cytokine. Conclusions: These findings show that HrIL-1β interacts with its receptor on smooth muscle cells to induce transcription of the IL-6 gene and to cause the secretion of IL-6. These results indicate that intestinal smooth muscle cells are not only targets for but also a source of cytokine.

Sympathetic nerve contact causes maturation of mast cells in vitro

Using a tissue culture model developed to study interactions between peripheral neurons and mast cells (MC), time-lapse microscopy showed that RBL-2H3 cells (a model of the mucosal MC) formed attachments with sympathetic neurons, ceased to divide, and moved along neurites toward the cell bodies. Electron microscopy showed significant increase in granules compared to intrinsic controls (RBL cells in coculture but lacking neurite contact). In studies using cohort cultures of 12- to 14-day-old sympathetic neurons, RBL cells adhered more rapidly to neurons than did control YB2/0 cells (a neutral target cell), and were inhibited in growth compared with RBL cells cultured in parallel without neurons. RBL cells cocultured with neurons for 24-48 h took up significantly more 3H-5HT and released a significantly larger percentage of 3H-5HT in response to the calcium ionophore A23187 than RBL cells in parallel pure cultures. Since no change in MC phenotype was seen, we conclude that contact with nerve membrane may be a developmental cue leading to maturation of MC.

Lysophosphatidylserine potentiates nerve growth factor-induced differentiation of PC12 cells

Since lysophosphatidylserine (LPS) is required for nerve growth factor (NGF)-induced secretion of histamine from rat mast cells, we investigated whether LPS might potentiate the effects of NGF in inducing neural differentiation of PC12 cells. Cell morphology was evaluated 48 h after addition of NGF, LPS or NGF + LPS. LPS alone was ineffective, but strongly promoted NGF-induced differentiation to give rise to cells that more closely resembled neurons in primary culture. LPS increased the number of PC12 cells that developed neurites in response to NGF (0.01-40 ng/ml), with the response to 1.0 ng/ml increasing from 17.8 +/- 2.2 to 50.8 +/- 4.1% when LPS was also present. Neurite length was also greater in PC12 cells receiving NGF + LPS: 17.8 +/- 2.2% of cells had neurites longer than three cell body diameters with 1.0 ng/ml NGF + 1 microg/ml LPS, compared to 1.6 +/- 1.6% with NGF alone. Further, cells responding to NGF + LPS typically developed only 1-2 neurites per cell (90.9%, 1 microg/ml LPS), compared with the multipolar appearance with NGF alone (71.1% with 3-6 neurites, 10 ng/ml NGF). LPS occurs at sites of tissue damage where NGF can also be present, and therefore may be a naturally-occurring modifier of neuronal structure and/or function.

Apparent innervation of rat basophilic leukaemia (RBL-2H3) cells by sympathetic neurons in vitro

Association of mast cells (MCs) and nerves may represent communication between the immune and nervous systems. The morphological features of associations between sympathetic neurons and rat basophilic leukaemia cells (RBL), a model for the mucosal mast cell, were studied in a tissue culture model. Initially, neuronal growth cones contacted single RBL with large areas of membrane apposition. With time, these appeared as distinct zones of contact where intervening distances were less than or equal to 50 nm. Large dense-cored granules suggested localization of peptidergic neurotransmitters. Encircling of neurite profiles by RBL resembled intimate nerve-MC relationships in vivo. These modifications may serve to optimize the area of interaction.

Smooth muscle from aganglionic bowel in Hirschsprung's disease impairs neuronal development in vitro

Hirschsprungs disease results from the congenital absence of enteric neurons in human distal colon. The reason for aganglionosis is unknown but may reflect an unfavourable microenvironment for neuronal development. We asked if smooth muscle cells from the aganglionic region could affect neuronal development in vitro. Neurons from neonatal mouse superior cervical ganglia were added to cultures of smooth muscle obtained from normal or aganglionic regions of five patients with Hirschsprungs disease. Although neurons initially showed more rapid attachment to aganglionic smooth muscle, this was equal by 60 min and thereafter. Progressive increase in the diameter of the nerve cell body was characteristic of normal maturation in vitro. This was consistently inhibited by 15–22% in neurons grown on aganglionic muscle compared with normal controls over the 6-day test period (P<0.05). This phenomenon was preserved when the smooth muscle cells were lysed by brief exposure to distilled water before initiation of coculture (16–18% inhibition; P<0.05). These data imply that smooth muscle of the aganglionic colon is less favourable for neuronal development than the normally innervated region and suggest a membrane-linked factor. Clearly, this persists in postnatal life and in vitro and may reflect an abnormality of cellular interaction causing Hirschsprungs disease.