Bi-directional Relationship of in Vitro Mast Cell-Nerve Communication Observed by Confocal Laser Scanning Microscopy.

Abstract

During the last decade there has been an exponential increase in data illustrating that the immune and nervous systems are not disparate entities.1,2) The mast cell-nerve relationship has served as a prototypic association and has provided substantial evidence for bi-directional communication between nerves and immune cells.3) Early studies elegantly described the non-random spatial association of mast cells and nerves in a variety of tissues in which actual membranemembrane contacts could be observed. 4,5) To understand these events, we have recently studied direct neurite-mast cell communication using an in vitro co-culture approach and calcium imaging by confocal laser scanning microscopy (CLSM). Our results showed clearly that nervemast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating via NK-1 receptors, is an important mediator of this communication.6) In addition, we studied direct nervemast cell communication by atomic force microscopy (AFM). AFM showed precisely cell surface structures in which neurites attached to a pseudopodium and a cell body of a mast cell. 7) Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airway inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature. 6,7) However, the mast cell-nerve interaction is not a one-sided relationship but a bi-directional one.3) The consequent effects of such potentially reverberating circuits on local physiology may have great significance in the initiation or perpetuation of disease states, such as bronchial hyperreactivity and asthma, idiopathic functional bowel disorders, food allergy, and eczema.In the present work, we have studied in more detail the communication from mast cells to neurites. Our results clearly show that communication from mast cells to neurites exists in the in vitro nerve-mast cell co-culture system. MATERIALS AND METHODSNerve-Mast Cell Co-culture Following a published protocol, superior cervical ganglia (SCG) were dissected from newborn (0-48 h old) CBA mice (Japan SLC, Shizuoka, Japan) and rinsed in Hanks' balanced salt solution containing 10 mM HEPES (pH 7.4). Each ganglion was divided into two to four pieces and incubated for 60 min at 37°C in 2 ml of HEPES containing 0.125% trypsin (grade II; Sigma, St. Louis, MO, U.S.A.). The resultant cell suspension was plated at a density of 0.5-1ϫ10 4 nerve cells onto matrigel (Becton Dickinson, Bedford, MA, U.S.A.)-coated 35-mm diameter glass dishes. The neurons were grown in F12 culture medium (Life Technologies, Rockville, MD, U.S.A.) supplemented with 0.2 mM L-glutamine, 0.3% glucose, 3% antibiotic/antimycotic (A-7292) (all from Sigma), 10% FBS (BioWhittaker, Walkersville, MD, U.S.A.), and 50 ng/ml murine nerve growth factor (NGF, 2.5 S; Upstate Biotechnology, Lake placid, NY, U.S.A.). Nonganglionic cells were killed by an initial exposure to cytosine-b -D-arabinofuranoside (Ara-C, 10 Ϫ6 M; Sigma) for 24 h. Further deta...

Nerve-mast cell (RBL) interaction: RBL membrane ruffling occurs at the contact site with an activated neurite

“…Using the murine SCG-RBL cell system as a model of neuron-mast cell association, we showed bidirectional communication between the cells by using either specific neuronal [BK or SV (21)] or mast cell [i.e., anti-IgE antibodies (22)] activators as the initiating stimuli. These studies depended largely on Ca 2ϩ imaging as the index of cell activity.…”

N-Cadherin Plays a Role in the Synapse-Like Structures between Mast Cells and Neurites

“…[6][7][8] In addition we studied by atomic force microscopy (AFM) the morphological structure of the interaction between neurites and mast cells (RBL) which was involved in the communication.…”

“…6) Our findings have implications for the neuroimmune signaling cascade that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in which mast cells feature. [6][7][8] In addition we studied by atomic force microscopy (AFM) the morphological structure of the interaction between neurites and mast cells (RBL) which was involved in the communication. 9) AFM images showed that association between the growth cone of neurites and the RBL cell occurred over ca.…”

Molecular Basis of Neuroimmune Interaction in an In Vitro Coculture Approach

“…Our data illustrated that SCG neurites were activated in response to RBL activation after a lag time (Figure 2) (61,73). In coculture dishes, anti-IgE antibody-or antigen-induced RBL activation was invariably followed by SCG neurite activation.…”