Secretion of Mast Cell Inflammatory Mediators Is Enhanced by CADM1-Dependent Adhesion to Sensory Neurons

Abstract

Neuroimmune interactions are important in the pathophysiology of many chronic inflammatory diseases, particularly those associated with alterations in sensory processing and pain. Mast cells and sensory neuron nerve endings are found in areas of the body exposed to the external environment, both are specialized to sense potential damage by injury or pathogens and signal to the immune system and nervous system, respectively, to elicit protective responses. Cell adhesion molecule 1 (CADM1), also known as SynCAM1, has previously been identified as an adhesion molecule which may couple mast cells to sensory neurons however, whether this molecule exerts a functional as well as structural role in neuroimmune cross-talk is unknown. Here we show, using a newly developed in vitro co-culture system consisting of murine bone marrow derived mast cells (BMMC) and adult sensory neurons isolated from dorsal root ganglions (DRG), that CADM1 is expressed in mast cells and adult sensory neurons and mediates strong adhesion between the two cell types. Non-neuronal cells in the DRG cultures did not express CADM1, and mast cells did not adhere to them. The interaction of BMMCs with sensory neurons was found to induce mast cell degranulation and IL-6 secretion and to enhance responses to antigen stimulation and activation of FcεRI receptors. Secretion of TNFα in contrast was not affected, nor was secretion evoked by compound 48/80. Co-cultures of BMMCs with HEK 293 cells, which also express CADM1, while also leading to adhesion did not replicate the effects of sensory neurons on mast cells, indicative of a neuron-specific interaction. Application of a CADM1 blocking peptide or knockdown of CADM1 in BMMCs significantly decreased BMMC attachment to sensory neurites and abolished the enhanced secretory responses of mast cells. In conclusion, CADM1 is necessary and sufficient to drive mast cell-sensory neuron adhesion and promote the development of a microenvironment in which neurons enhance mast cell responsiveness to antigen, this interaction could explain why the incidence of painful neuroinflammatory disorders such as irritable bowel syndrome (IBS) are increased in atopic patients.

Keywords: CADM1; IGE receptor; allergy; mast cells; pain; sensory neurons; synCAM1.

FIGURE 1

CADM1 is expressed in BMMCs and sensory neurons. (A) Immunoblot of lysates prepared from DRG and BMMC mono-cultures probed with anti-CADM1 and anti-α-Tubulin, as a loading control. The m.w. scale is shown to the right of the blot. (B) Flow cytometric analysis of surface (left) and total CADM1 expression in Triton X-100-permeablized BMMCs (right). BMMCs were double labeled with conjugated c-Kit and CADM1 Abs. BMMCs were gated as c-kit+ CADM1high cell subset and c-kit+ CADM1medium cell subset. Numbers in plots are the percentages of cells in the indicated gate. (C) Confocal immunofluorescence images of c-Kit (red), CADM1 (green), and DAPI (blue) and merged image of mono-cultured BMMCs. Scale bar represents 10 μm. (D,E) Confocal immunofluorescence images of β-Tubulin (red), CADM1 (green) and DAPI (blue) in mono-cultured DRGs. White arrows indicate CADM1 immunoreactive neurites. Note that non-neuronal cells, observed as DAPI positive, β-Tubulin negative are also negative for CADM1 (arrowheads). Scale bar represents 20 μm.

FIGURE 2

CADM1 is enriched at mast cell-sensory neurite contact sites. Representative immunofluorescent images of BMMCs co-cultured with DRG neurons for 24 h. In panel (A) individual and overlaid images are shown from a neurite with two adherent cells. β-Tubulin (red) is used as the neuronal marker, DAPI (blue) nuclear marker labels all cells in the culture, while FcεRI (yellow) specifically labels BMMCs. CADM1 (green) stained positive in the sensory neuron neurite and BMMC, but is absent from the attached non-neuronal, non-mast cell. (B,C) Representative individual and overlaid immunofluorescent images of CADM1 (green), β-Tubulin (red) and DAPI (blue) staining of different co-cultures. Increased CADM1 fluorescent intensity at contact sites (white arrows) between BMMCs and neurites (red).

FIGURE 3

Adhesion of mast cells to sensory neurons is attenuated by a CADM1 blocking peptide. (A) Superimposed bright-field and fluorescent image of live calcein-labeled, adherent BMMCs (green) co-cultured with DRG neurons (unlabeled cells) for 2 h. Non-adherent cells have been removed by washing and centrifugation of the plate. (B) Image of calcein-labeled BMMCs plated in parallel into matrigel-coated wells devoid of neurons and subjected to the same washing and centrifugation procedure. (C) BMMC adhesion quantified from calcein-fluorescence remaining in wells after washing and centrifugation expressed as a percentage of total well fluorescence measured prior to washing procedure. Data shown as mean ± SEM from N = 3. Each done in duplicate. Data were analyzed using a two-tailed paired t-test ^∗∗p < 0.01. (D) Concentration-dependent inhibition of mast cell adhesion to DRG measured with a CADM1 blocking peptide. Percentage of adherent BMMC was calculated using the calcein adhesion assay. Each condition was done in duplicate, on N = 3 cultures. Each point represents the mean ± SEM. One-way ANOVA followed by Turkey’s multiple comparison post-test was performed. ^∗∗ denotes p < 0.01 and ^∗∗∗ p < 0.001 compared to the percentage of BMMC adhesion in the absence of CADM1 blocking peptide.

FIGURE 4

Knockdown of CADM1 in mast cells inhibits their adhesion to sensory neurons. (A) Western blot analysis of CADM1 expression in BMMCs transfected with the indicated ShRNA 48 h previously. Expression from un-transfected control cells examined in parallel are also shown (lane labeled BMMC). GAPDH was used as loading control. Data are representative of three independent experiments. (B) Adherence of BMMCs transfected with CADM1 ShRNA or scrambled control to neurons tested using the calcein assay. BMMC adhesion in the absence of DRG neurons is shown as a control. CADM1 knockdown significantly reduced BMMC adhesion to sensory neurons (N = 3). Each bar represents the mean ± SEM. One-way ANOVA followed by Turkey’s multiple comparison post-test was performed. ^∗∗∗ denotes p < 0.001.

FIGURE 5

Co-culture of BMMCs with DRG does not impact neuronal morphology. Representative schematic tracings of the neurites from DRG cultured alone (A) or in BMMC-DRG co-culture (B). Soma and neurites were visualized with anti-β-III tubulin (red) and BMMCs with anti-c-kit (green, white arrows) in DRG mono-cultures (C) and co-cultures (D). DAPI staining of nuclei (blue) show presence of non-neuronal cells in the DRG cultures. (E) Quantitative analysis of total neurites length in DRG mono-cultures and co-cultures, compared using unpaired t-test. (F) Sholl analysis of the neurite complexity. Each bar represents the mean ± SEM of number of crossing neurites found in each given distance from the soma. Statistical analysis using multiple t-test. N = 3.

FIGURE 6

Mast cell degranulation is potentiated following co-culture with sensory neurons. BMMCs pre-sensitized with anti-DNP IGE were cultured alone or with DRG for various times as indicated (A–F). Degranulation measured as secreted β-hexosaminidase (β-hex) in media recovered from the cultures was measured at the times indicated above each graph in the absence (A–C) or after 30 min exposure to the antigen (Ag) DNP (10°ng/ml, D–F) and expressed as a percentage of total β-hex measured from BMMC lysed with 0.5% Triton X-100. Data shown are mean ± SEM of N = 3, each performed in duplicate. ^∗p < 0.05, ^∗∗ p < 0.01 and ^∗∗∗ p < 0.001 compared to BMMC alone. Data were analyzed using two-tailed paired t-test.

FIGURE 7

Potentiation of mast cell degranulation by sensory neurons is contact-dependent. (A) BMMC degranulation measured as secreted β-hex from the mono-cultures and co-cultures of the indicated cells. Sensory neurons co-cultured with BMMCs for 24 h, were activated with 1 μm capsaicin for 20 min. β-Hex secreted from mono-cultures of DRGs is minimal and shows that the potentiated BMMC degranulation observed in the co-cultures is not simply additive but due to a significant interaction between the two cell types. (B) BMMCs pre-sensitized with anti-DNP IGE were cultured alone or with supernatant collected from BMMC-DRG co-cultured for 24 h. Degranulation as measured by secreted β-hex was measured in resting condition (labeled Ag independent) or after 30 min stimulation with the antigen, DNP (10 ng/ml, labeled Ag-mediated), and expressed as a percentage of total β-hex measured from lysed BMMCs. (C) Pre-sensitized BMMCs were cultured alone or on the top of DRG cultured using a transwell system for 24 h prior to measuring degranulation. Data shown are mean ± SEM of N = 3, each performed in duplicate. Data were analyzed using two-tailed paired t-test. ^∗p < 0.05, ^∗∗p < 0.01, n.s is non-significant.

FIGURE 8

CADM1 is necessary for mast-cell sensor neuron cross-talk. BMMCs pre-sensitized with anti-DNP IGE were co-cultured with DRG for 24 h in the presence or absence of 10 μg/ml CADM1 blocking peptide before degranulation was quantified using the β-Hex assay. Data were normalized to degranulation measurements made from BMMC mono-cultures set up in parallel, and the fold-change in basal Ag-Indep degranulation (A) or DNP (10 ng/ml), Ag-activated degranulation (B) calculated and compared by two-tailed paired t-test, ^∗ denotes p < 0.05, ^∗∗p < 0.01. Each bar represents the mean ± SEM (N = 3). (C) Untransfected (WT BMMC), CADM1-ShRNA or scramble-transfected BMMCs pre-sensitized with anti-DNP IGE were co-cultured with DRG for 24 h before degranulation was quantified using the β-Hex assay. Fold change of Ag-Independent degranulation (C) and DNP (10 ng/ml) Ag-activated degranulation (D) were calculated and compared by one-way ANOVA followed by Turkey’s multiple comparison post-test, ^∗∗∗denotes p < 0.001 compared to wt BMMC-DRG co-culture. Each bar represents the mean ± SEM (N = 3).

FIGURE 9

Sensory neuron potentiation of antigen-stimulated cytokine secretion by mast cells is dependent on CADM1. BMMCs pre-sensitized with anti-DNP IGE were cultured alone or with DRG for 24 h. (A) Secreted IL-6 and (B) TNFα were quantified by ELISA for cells stimulated in parallel with the antigen DNP (10 ng/ml) for 6 h or vehicle control. Data shown are mean ± SEM of N = 3, each performed in duplicate. ^∗ p < 0.05 compared to stimulated BMMC alone. Data were analyzed using two-tailed paired t-test. (C) BMMC mono-culture and untransfected WT, CADM1-knockdown or scramble-transfected BMMCs were co-cultured with DRG for 24 h, prior to being activated with DNP (10 ng/ml) for 6 h. IL-6 was measured from supernatants and compared by one-way ANOVA followed by Turkey’s multiple comparison post-test, ^∗denotes p < 0.05 compared to wt BMMC-DRG co-culture. Each bar represents the mean ± SEM (N = 3).