MRGPR-X1 induce CCR2 via NFAT in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 6 h (A-C) using specific primers for 5 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (B and C) cells were treated or not with CsA (1 µM, 30 min). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. BAM8-22-induced (1 µM, 20 h) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by flow cytometry (D) or by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation (E). In (D) one representative experiment is shown. Accumulation of the data from 5 independent experiments revealed an increase in the number of CCR2 positive cells by 10.8±1.4% after BAM8-22 stimulation. In (E, left panel) one representative experiment is shown and in (E, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1 stimulate TCF/SRF- and NFAT-dependent reporter in HEK293- and F11-MRGPR-X1 cells.

<p>TCF/SRF-, NFAT- or CREB-dependent reporter gene constructs were transfected into HEK293-MRGPR-X1 (A–C) or in F11-MRGPR-X1 (D–F) cells. Cells were stimulated with 1 µM BAM8-22 for 6 h or as indicated. 5 µM FSK was used as a control for the CREB reporter. CsA (1 µM, 30 min) was used to block the NFAT activator calcineurin and PD-184352 (10 µM, 30 min) to block ERK-1/2 activity. Data from 3–5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate significant differences to not stimulated cells. Dagger signs indicate significant differences between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1 induce EGR-1 via ERK-1/2 in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 1 h (A, C and D) using specific primers for 15 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (C and D) cells were treated or not with PD-184352 (30 min, 10 µM). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. In (B) serum-starved F11-MRGPR-X1 cells were stimulated or not with BAM8-22 (1 µM) for the indicated period of time and expression of EGR-1 was determined by western-blotting. Afterwards blots were stripped and re-probed with an antibody against ERK-2 (t-ERK-2). One representative blot is shown. Ligand-induced EGR-1 expression was quantified by densitometry and is given normalized to not stimulated cells. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisk indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

MRGPR-X1 induce EGR-1 via ERK-1/2 and CCR2 via NFAT in primary DRG neurons.

<p>BAM8-22-induced (2 µM) calcium signals in rat DRG neurons co-expressing MRGPR-X1 and aequorin or solely aequorin are presented in (A). BAM8-22-induced (2 µM, 8 h) activation of the NFAT (B) or TCF/SRF (C) reporter is shown in rat DRG neurons transiently co-expressing MRGPR-X1. RTQ-PCR experiments were performed with cDNAs derived from serum-starved MRGPR-X1 expressing rat DRG neurons stimulated or not with BAM8-22 (2 µM) for 6 h (D) or 40 min (E). CsA (1 µM, 30 min) was used to block calcineurin in (B and D) or PD-184352 (10 µM, 30 min) to inhibit ERK-1/2 activity in (C and E). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

B2R do not induce CCR2 despite NFAT activation.

<p>(A) BAM8-22- (2 µM) or BK-induced (1 µM) calcium signals were determined in single fura2-loaded F11-MRGPR-X1 cells by calcium imaging. Data of ∼300 cells were compiled and expressed as the mean ± S.E.M. RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BK (1 µM) for 1 h in (B) or 6 h in (E). Relative BK-induced gene expression was normalized to β-actin, calculated using the ΔΔCp method, and expressed as the mean ± S.E.M. (C) Serum-starved F11-MRGPR-X1 cells were stimulated or not with BK (1 µM) for the indicated period of time and expression of EGR-1 was determined by western-blotting. Afterwards blots were stripped and re-probed with an antibody against ERK-2 (t-ERK-2). One representative blot is shown. Ligand-induced EGR-1 expression was quantified by densitometry and is given normalized to not stimulated cells as the mean ± S.E.M. (D) BK-induced (1 µM, 6 h) activation of the NFAT reporter is shown in F11-MRGPR-X1 cells. Data are expressed as the mean ± S.E.M. PD-184352 (10 µM, 30 min) was used to inhibit ERK-1/2 activity in (B) or CsA (1 µM, 30 min) to block calcineurin in (D and E). In (B–E) 4 independent experiments were conducted, respectively. In (F) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation after pre-stimulation of the cells with BAM8-22 or BK (1 µM, 20 h). In (F, left panel) one representative experiment is shown and in (F, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BK-stimulated inhibitor-treated and untreated cells or between BK- and BAM8-22-stimulated cells.</p

MRGPR-X1-induced signaling in DRG neurons and CTMCs.

<p>A cartoon illustrating the signaling circuit by which MRGPR-X1 affect chemokine signaling in DRG neurons and connective tissue mast cells is given.</p

MRGPR-X1-induced CCL2 release in LAD-2 mast cells.

<p>(A) MRGPR-X1 (40 cycles) or β-actin (30 cycles) mRNA expression was determined in LAD-2 cells by RT-PCR. As a negative control, RT-PCR was conducted without addition of cDNA (H₂O). (B) Calcium signals in fura2-loaded LAD-2 cells are shown after injection of BAM8-22 (5 µM) or ionomycin (5 µM) or HBS as positive or negative control, respectively. (C) CCL2 release in LAD-2 cells after stimulation with BAM8-22 (5 µM, 18 h) was determined by ELISA. Data from 4 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells.</p

Overlapping AQP4 peptides.

<p>Overlapping AQP4 peptides.</p

Functional characterization of AQP4_22–36 peptide specific TCL8.2.

<p>Representative T cell line (TCL) reactive to AQP4_22–36. (A) TCL8.2 cells were restimulated with irradiated syngeneic splenocytes in the presence of increasing concentrations of AQP4_22–36. The proliferative response was determined by ³[H] thymidine incorporation. Mean c.p.m. of triplicate cultures + SD. (B) Cytokine production of TCL8.2 cells in response to increasing concentrations of AQP4_22–36 as measured by cytometric bead array in the cell culture supernatant collected at 48 h after initiation of restimulation. Mean cytokine concentrations + SD of triplicate cultures. (C) Intracellular cytokine staining of TCL8.2 after 6 restimulation cycles.</p

Antibody response to AQP4.

<p>Wild type C57BL/6 mice were immunized with full length AQP4/CFA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016083#s4" target="_blank">Materials and Methods</a>. Unimmunized mice or mice immunized with MOG_35–55/CFA were used as controls. Sera of mice from each group were collected and tested for AQP4 specific antibodies in a cell based flow cytometry analysis. Subclass specification was performed by using fluorochrome labeled anti-mouse Ig antibodies specific for IgM, IgA, IgE, IgG1, IgG2a, IgG2b, and IgG3 (FITC labeled) or total IgG H+L (AlexaFluor488 labeled). (A) Representative histogram plots illustrating the MFIs for AQP4 specific IgG H+L in the various test groups. (B) Representative histogram plots of AQP4 specific Ig classes and IgG subclasses in AQP4 immunized animals. (C) ΔMFIs + SEM (n = 4) for AQP4 specific IgG H+L in naive control mice, MOG_35–55 immunized mice, or full length AQP4 immunized mice. (D) ΔMFIs + SEM (n = 4) for individual anti-AQP4 Ig classes and IgG subclasses in full length AQP4 immunized mice.</p