Figure 1.
5-HT induces rolling of human Eos and alters cell shape and morphology.
(A) Expression of 5-HT receptors by human Eos from allergic donors (D1-D5) by RT-PCR. Expression of β-actin is shown as the internal control. (B) Rolling of human Eos on rh VCAM-1-coated cover-slips under conditions of flow in vitro. Cells were treated with 5-HT or the 5-HT2A/2C receptor agonist DOI (both at 10 µM) for 5 min prior to infusion into the flow chamber. Cells treated with DMSO (vehicle) alone served as a control. (C) FSC versus side scatter (SSC) dot plots for untreated (control) and 5-HT (10 µM)- or C5a (1 nM, positive control)-treated human Eos after 1 min by GAFS assay. (D) Human Eos shape change measured as an increase in FSC after treatment with 5-HT or C5a for 10 min at 37°C measured. Results are expressed as percent change in mean FSC of stimulated cells compared to control cells. (E) Confocal microscopy of FITC-phalloidin stained human Eos initially allowed to attach to rh VCAM-1 on cover-slips for 20 min in PBS followed by an additional 5 min in the presence of 5-HT or DOI (both at 10 µM) or vehicle. Magnification ×600. Combined data (Mean ± SEM) of Eos from 4–6 donors is shown for B and D. *p<0.01 for 5-HT and <0.03 for DOI in B for comparison with DMSO-treated Eos and <0.01 in D for comparison with control Eos. Representative data of six independent experiments in C and three independent experiments in E with Eos from different donors is shown.
Figure 2.
5-HT and DOI induce rolling of AML14.3D10 human Eos-like cells.
(A) Expression of 5-HT2A by AML14.3D10 cells by RT-PCR with β-actin as the internal control. (B) Rolling of AML14.3D10 cells on rh VCAM-1-coated cover-slips under conditions of flow in vitro after treatment with 5-HT, DOI or DMSO (vehicle) for 5 min prior to infusion into flow chamber. (C) Confocal microscopy of FITC-phalloidin stained AML14.3D10 cells adhered to rh VCAM-1 on cover-slips in the presence of vehicle (DMSO), 5-HT or DOI as described in Figure 1. Magnification ×600. Combined data (Mean ± SEM) of 3 (for 5-HT)-5 (for DOI) independent experiments in duplicate is shown for B. *p<0.03 in B for comparison of 5-HT- or DOI-treated versus vehicle-treated cells. Data shown in C is representative of three independent experiments.
Figure 3.
DOI-induced rolling of AML14.3D10 cells involves activation of ROCK, MAPK PI3K, PKC and calmodulin.
(A) Phosphorylation of p-44/42 MAPK in cells exposed to DOI after pre-treatment with PD98059 (10 µM, MAPK inhibitor) or DMSO (vehicle for inhibitor) for 5 min. Cell lysates were analyzed by Western blot analysis. (B) DOI-induced rolling of AML14.3D10 cells on rh VCAM-1-coated cover-slips under conditions of flow in vitro after treatment with MDL-100907 (5-HT2A antagonist), Y27632 (ROCK inhibitor), PD98059, LY294002 (PI3K inhibitor) (left panel) or PT (Gαi inhibitor), BIM(I) (PKC inhibitor), TFP (calmodulin inhibitor) (right panel). Cells treated with DMSO alone served as the control. All inhibitors were used at a concentration of 10 µM, except PT which was used at 100 ng/ml, for 20 min and then exposed to DOI for 5 min prior to infusion into the flow chamber. (C) Total F-actin measured by flow cytometry after FITC-phalloidin staining of AML14.3D10 cells treated with MDL-100907, Y27632, PD98059, LY294002 or vehicle for 20 min and then exposed to DOI. Results are expressed as percent change in mean fluorescence intensity (MFI) relative to vehicle-treated cells. (D) FITC-phalloidin staining and confocal microscopy of AML14.3D10 cells adhered to rh VCAM-1-coated cover-slips in the presence of MDL-100907, Y27632, PD98059, LY294002 or vehicle and then exposed to DOI (as described in Figure 1). Magnification ×600. Combined data (Mean ± SEM) of four independent experiments in B and three independent experiments in C is shown. *p<0.03 for comparison of cells exposed to DOI versus vehicle. **p<0.01 and #p<0.05 for comparison of cells exposed to DOI in the presence of inhibitor versus cells exposed to DOI in the presence of vehicle. Data shown in D is representative of two independent experiments.
Figure 4.
Effect of 5-HT and DOI on murine Eos rolling in vivo and in vitro.
(A) Expression of 5-HT2 receptor subtypes by murine BM-Eos by RT-PCR. Expression of β-actin is shown as a control. (B) Trafficking of infused murine BM-Eos in inflamed (TNFα-stimulated) cremaster muscle microvessels of anesthetized mice before and after superfusion with 5-HT (100 nM) by IVM. The number of rolling cells is expressed as rolling fraction which is a percentage of the total number of cells passing through the same reference point. Rolling velocity of interacting Eos determined by off-line analysis of recorded video images by choosing four to six rolling Eos per venule and measuring the time taken for the cells to travel between two reference points (50-200 μm). Results represent mean rolling velocity of 143 cells before 5-HT treatment and 164 cells after 5-HT treatment. Number of Eos adhered in cremaster muscle microvessels of mice before and after treatment with 5-HT is also shown. An average of 16±2 recorded video images with 1–2 vessels per field were analyzed per mouse. Combined data (Mean ± SEM) of n = 3 mice/group is shown. *p<0.01 for comparison of Eos trafficking (rolling, rolling velocity and adhesion) before versus after 5HT. (C) Rolling of murine Eos on rm VCAM-1-coated cover-slips under conditions of flow in vitro after treatment with MDL-100907, Y27632, PD98059, LY294002 (left panel), BIM(I), TFP, PT (right panel) or DMSO (vehicle) as described in Figure 3. Combined data (Mean ± SEM) of 3 (right panel) –4 (left panel) independent experiments is shown. *p<0.01 for comparison of cells exposed to DOI versus vehicle. **p<0.01 and #p<0.02 for comparison of cells exposed to DOI in the presence of inhibitor versus cells exposed to DOI in the presence of vehicle.
Figure 5.
DOI induces changes in morphology of murine Eos.
Effect of (A) MDL-100907, Y27632, PD98059, LY294002 as well as (B) PT, BIM(I) and TFP on DOI-induced changes in morphology of murine Eos adherent on rm VCAM-1 by confocal microscopy. Mouse Eos were allowed to adhere to rm VCAM-1-coated cover-slips in the presence of inhibitors or DMSO (vehicle) for 20 min and then exposed to 10 μM DOI for an additional 5 min prior to staining with FITC-phalloidin. Cells were counter-stained with DAPI. Magnification ×400. Quantitation of changes in morphology of murine Eos adherent on rm VCAM-1 exposed to DOI in the absence or presence of (C) MDL-100907, Y27632, PD98059, LY294002 as well as (D) PT, BIM(I) and TFP. Adhered cells in a fixed number of randomly selected non-overlapping fields of each cover-slip were counted and cells exhibiting cell spreading and/or distinct leading edges were identified and expressed as a percentage of the total number of cells in the field. Data shown in A and B is representative of three independent experiments with Eos from three different mice. Combined data (Mean ± SEM) from three independent experiments in C and two independent experiments in D in duplicate is shown. *p<0.05 and **p<0.01 for comparison of cells exposed to DOI in the presence of inhibitor versus cells exposed to DOI in the presence of vehicle.
Figure 6.
5HT and DOI induce migration of murine Eos.
(A) Migration of murine Eos towards 10 μM 5-HT or DOI in Transwell® plates after 4 h at 37°C. The average number of cells/field/well was determined and results expressed as a percentage of background migration observed in wells containing medium alone. Combined data (Mean ± SEM) of 5 independent experiments is shown. *p<0.01 for comparison of 5-HT- or DOI-treated cells versus vehicle-treated cells. (B) Effect of MDL-100907, Y27632, PD98059, LY294002, BIM(I), TFP (all at 10 μM), and PT (100 ng/ml) on DOI-induced migration of murine Eos. Cells were pre-treated with inhibitors or DMSO (vehicle) alone for 20 min before addition to Transwell® Chambers. Results are expressed as a percentage of the migration of vehicle treated cells towards DOI. Combined data (Mean ± SEM) of 3 (for PT, BIM(I), TFP) or 5 (all other inhibitors) independent experiments in duplicate or triplicate is shown. *p<0.01 and **p<0.02 for comparison of cells treated with inhibitors versus cells treated with vehicle. (C) Basal and DOI-induced [Ca2+]i levels in murine Eos from 364 cells by digital videofluorescence imaging with Fura-2 AM. Representative data of three independent experiments performed in triplicate. *p<0.01 compared to unstimulated cells. (D) Expression of adhesion molecules by murine Eos after treatment with 10 μM DOI (or PBS) for 5 min by flow cytometry using rat mAbs against α4 (CD49), Mac-1 (CD11b) and LFA-1 (CD11a) followed by FITC-conjugated goat anti-rat IgG as the secondary antibody. Depending on the mAb, rat IgG2a or 2b was used as the isotype matched control. Expression of CD62L was evaluated using FITC-conjugated anti-mouse CD62L (BD Biosciences). FITC-conjugated rat-IgG2a was used as the isotype control for CD62L. All antibodies were used at a final concentration of 5 μg/ml. Data shown is representative of three independent experiments with Eos from different mice.