Mast cells can secrete vascular permeability factor/ vascular endothelial cell growth factor and exhibit enhanced release after immunoglobulin E-dependent upregulation of fc epsilon receptor I expression

Abstract

Vascular permeability factor/vascular endothelial cell growth factor (VPF/VEGF) can both potently enhance vascular permeability and induce proliferation of vascular endothelial cells. We report here that mouse or human mast cells can produce and secrete VPF/VEGF. Mouse mast cells release VPF/VEGF upon stimulation through Fcepsilon receptor I (FcepsilonRI) or c-kit, or after challenge with the protein kinase C activator, phorbol myristate acetate, or the calcium ionophore, A23187; such mast cells can rapidly release VPF/VEGF, apparently from a preformed pool, and can then sustain release by secreting newly synthesized protein. Notably, the Fc epsilonRI-dependent secretion of VPF/VEGF by either mouse or human mast cells can be significantly increased in cells which have undergone upregulation of Fc epsilonRI surface expression by a 4-d preincubation with immunoglobulin E. These findings establish that at least one cell type, the mast cell, can be stimulated to secrete VPF/VEGF upon immunologically specific activation via a member of the multichain immune recognition receptor family. Our observations also identify a new mechanism by which mast cells can contribute to enhanced vascular permeability and/or angiogenesis, in both allergic diseases and other settings.

Figure 1

Northern blot analysis of total RNA from C1.MC/C57.1 mast cells stimulated through FcεRI (A) or with PMA (B). C1.MC/ C57.1 cells were sensitized with IgE (10 μg IgE/ml) for 30 min, washed, and then stimulated with either 50 ng/ml DNP-HSA or 50 ng/ml PMA. Control cells (at 0.5 and 24 h) were challenged with medium (in A) or vehicle (0.005% DMSO, in B). Blotted total RNA from cells harvested at various times after stimulation was probed for VPF/VEGF and G3PDH (to demonstrate equal RNA loading).

Figure 2

ISH studies of freshly isolated mouse PMCs. Expression of VPF/VEGF mRNA is higher in mast cells that had been treated with PMA (50 ng/ml) for 2 h before hybridization to an ³⁵S-labeled antisense riboprobe (A) than in vehicle-treated (control) mast cells that had been hybridized to the same antisense riboprobe (B). No specific signal is seen in PMA-stimulated mast cells hybridized to the ³⁵S-labeled sense (control) riboprobe (C  ). Bar = 16 μm. (D) Graphic depiction of VPF/VEGF expression by PMA- versus vehicle-treated mast cells.

Figure 3

Immunofluorescent detection of VPF/VEGF immunoreactivity in a highly purified (99% pure) population of freshly isolated mouse PMCs. (A and B) Little or no staining of PMCs that had been incubated for 2 h with vehicle alone (A) or PMA (50 ng/ml; B) before incubation with a control rabbit IgG preparation (IgG). (C and D) VPF/ VEGF immunoreactivity was detected with a rabbit anti-VPF/ VEGF (α-VEGF  ) antibody in some PMCs that had been incubated for 2 h with either vehicle alone (C  ) or PMA (50 ng/ml; D). Black bar in A (for A–D) = 50 μm. (E and F  ) Giemsa-stained cytospin preparations of the same vehicle- (E  ) or PMA- (F  ) stimulated purified PMCs that were used for immunofluorescent detection of immunoreactivity (A–D). Black bar in E (for E and F  ) = 50 μm.

Figure 4

Release of (A) VPF/VEGF (in 6 h) and (B) serotonin (5-HT, in 10 min) from BMCMCs that exhibit different levels of surface expression of FcεRI, after challenge with various concentrations of either specific antigen (DNP-HSA), the protein kinase C activator PMA, the calcium ionophore A23187, or SCF. BALB/c BMCMCs were cultured without IgE or with IgE at 5.0 μg/ml for 4 d before passive sensitization and antigen challenge or challenge with A23187, PMA, or SCF for 6 h. 0 in the PMA and A23187 experiments refers to cells incubated without PMA or A23187, respectively, but in the highest concentration of vehicle used for these studies (DMSO at 0.5%). All values are mean ± SEM (n = 8/point) of data pooled from two experiments with different batches of BMCMCs (each n = 4/point) that gave very similar results. *P < 0.05, **P < 0.005, or ***P < 0.0001 versus corresponding control values for unstimulated or vehicle-treated cells. ^† P < 0.05, ^†† P < 0.005, or ^††† P < 0.0001 versus corresponding values for cells cultured without IgE for 4 d. Note the different VPF/VEGF scale for PMA stimulation.

Figure 5

Kinetics of the release of VPF/VEGF from BALB/c BMCMCs that exhibit different levels of surface expression of FcεRI, after challenge with either specific antigen (DNP-HSA, at 50 ng/ml), PMA (50 ng/ml), A23187 (1 μM), or SCF (500 ng/ml). BMCMCs were cultured without IgE or with IgE at 5.0 μg/ml for 4 d before passive sensitization and antigen challenge or challenge with A23187, PMA, or SCF. Cells used as unstimulated controls for DNP-HSA– or SCF-stimulated cells were maintained in medium alone. PMA- or A23187-stimulated cells were compared with cells maintained in medium containing vehicle (DMSO at 0.1%). All values are mean ± SEM (n = 8/point) of data pooled from two experiments with different batches of BMCMCs (each n = 4/point) that gave very similar results. *P < 0.05, **P < 0.005, or ***P < 0.0001 versus corresponding control values for unstimulated or vehicle-treated cells. ^† P < 0.05, ^†† P < 0.005, or ^††† P < 0.0001 versus corresponding values for cells cultured without IgE for 4 d. Note the different VPF/VEGF scale for PMA.

Figure 6

Kinetics of release of 5-HT (A) and VPF/VEGF (B) from BMCMCs stimulated through the FcεRI. Mast cells were sensitized with IgE (10 μg IgE/ml) for 2 h and then preincubated as indicated for 5 min at 37°C with either no inhibitor, the inhibitor of intracellular protein transport Brefeldin A (5 μg/ ml), or the transcription inhibitor Act D (10 μg/ml). Cells were then challenged for various time periods with DNP-HSA (Antigen) or medium (Control  ). Data for VPF/VEGF are mean ± SEM (n = 3/point); data for [³H]5-HT are mean ± SEM (n = 4/point). *P < 0.05, ^† P < 0.005, ^‡ P < 0.0001 versus values for the same time point in cells incubated without the inhibitor, or, as indicated (brackets), versus corresponding values for control cells that were not challenged with DNP-HSA. A second identical experiment with BMCMCs gave very similar results.

Figure 7

IgE-induced enhancement of FcεRI expression in human mast cells increases the ability of these cells to release VPF/VEGF (over a 16-h period; A) and histamine (over a 1-h period; B) in response to FcεRI cross-linking. HMCs (12 wk of culture, 85% mast cell purity) were cultured without or with human myeloma IgE at 5 μg/ml for 4 d before further passive sensitization with IgE and challenge with goat anti–human IgE antibody. The data shown (mean ± SEM; n = 3/bar) are representative of the results obtained in three separate experiments using mast cells derived from three different cord blood donors.