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Immunoglobulin E induces VEGF production in mast cells and potentiates their pro-tumorigenic actions through a Fyn kinase-dependent mechanism.

Jiménez-Andrade GY, Ibarra-Sánchez A, González D, Lamas M, González-Espinosa C - J Hematol Oncol (2013)

Bottom Line: Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce.In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Farmacobiología, Cinvestav, IPN, Sede Sur, Calzada de los Tenorios 235, Col. Granjas Coapa, Tlalpan CP 14330, Mexico City, Mexico.

ABSTRACT

Background: High concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.

Methods: For in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE.In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn -/- mice (Wsh Rec Fyn -/-).

Results: Monomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn -/- BMMCs.

Conclusion: Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

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Monomeric IgE induces secretion of VEGF in BMMCs through a mechanism that requires Fyn. (A) Pharmacological characterization of IgE-induced VEGF secretion in MC. Two million BMMCs were pre-incubated with vehicle, Actinomicyn D (Act D; 5 μg/ml), Brefeldin A (BFA; 5μg/ml), Tetanus toxin (TTx; 10 ng/ml) and PP2 (10 μM) for 15 min in BMMC media. Then, cells were stimulated with 1000 ng of IgE for 8 h at 37°C. VEGF in cell supernatants was quantified by ELISA. (B) Role of different Src family kinases on IgE-induced VEGF secretion in MC. Two million BMMCs derived from WT, Fyn −/− and Lyn −/− mice were incubated with different amounts of IgE at 37°C for eight hours. Supernatants were collected and VEGF was determined by ELISA. (C) Time-course of VEGF secretion after the addition of IgE. Two million BMMCs were incubated in cell culture media containing 1000 ng/million cells of IgE at 37°C and supernatants were collected at different times after stimulation. VEGF was determined by ELISA. All results are shown as the mean ± SEM (n = 3-12). *, P < 0.05 compared with basal or time 0, &, P < 0.05 compared with IgE treatment, #, P < 0.05 compared to WT. One way ANOVA post hoc Student-Newman-Keuls (A). Two way ANOVA post hoc Student-Newman-Keuls (B and C).
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Figure 1: Monomeric IgE induces secretion of VEGF in BMMCs through a mechanism that requires Fyn. (A) Pharmacological characterization of IgE-induced VEGF secretion in MC. Two million BMMCs were pre-incubated with vehicle, Actinomicyn D (Act D; 5 μg/ml), Brefeldin A (BFA; 5μg/ml), Tetanus toxin (TTx; 10 ng/ml) and PP2 (10 μM) for 15 min in BMMC media. Then, cells were stimulated with 1000 ng of IgE for 8 h at 37°C. VEGF in cell supernatants was quantified by ELISA. (B) Role of different Src family kinases on IgE-induced VEGF secretion in MC. Two million BMMCs derived from WT, Fyn −/− and Lyn −/− mice were incubated with different amounts of IgE at 37°C for eight hours. Supernatants were collected and VEGF was determined by ELISA. (C) Time-course of VEGF secretion after the addition of IgE. Two million BMMCs were incubated in cell culture media containing 1000 ng/million cells of IgE at 37°C and supernatants were collected at different times after stimulation. VEGF was determined by ELISA. All results are shown as the mean ± SEM (n = 3-12). *, P < 0.05 compared with basal or time 0, &, P < 0.05 compared with IgE treatment, #, P < 0.05 compared to WT. One way ANOVA post hoc Student-Newman-Keuls (A). Two way ANOVA post hoc Student-Newman-Keuls (B and C).

Mentions: Production of angiogenic factors by MC has been shown to occur few hours after different stimuli, such as hypoxia, antigen or PMA [16,17]. To investigate if monomeric IgE in the absence of any antigen could induce VEGF secretion in this cell type, two million BMMCs were incubated with a monoclonal anti-DNP IgE (1000 ng/ml) for eight hours at 37°C in BMMC media. Supernatants were then collected and the amount of secreted VEGF was determined by ELISA. The addition of IgE to MC induced a significant secretion of VEGF (53.77 ± 2.15 pg/ml in basal conditions vs 117.16 ± 5.45 pg/ml on IgE-stimulated cells; Figure 1A). To gain insight on the mechanism involved in IgE-induced VEGF production, cells were pre-treated with different pharmacological inhibitors 15 minutes before the addition of IgE and secreted VEGF was measured. VEGF secretion was sensitive to actinomycin D (ActD) and brefeldin A (BFA), indicating that de novo transcription and transport from endoplasmic reticulum to the Golgi apparatus [18] was needed for IgE effects to occur. VEGF production was also affected by tetanus toxin (TTx), which inhibits secretion mediated by toxin-sensitive VAMPs (VAMP-1 and −2) [19], and PP2, that inhibits Src family kinases (Figure 1A).


Immunoglobulin E induces VEGF production in mast cells and potentiates their pro-tumorigenic actions through a Fyn kinase-dependent mechanism.

Jiménez-Andrade GY, Ibarra-Sánchez A, González D, Lamas M, González-Espinosa C - J Hematol Oncol (2013)

Monomeric IgE induces secretion of VEGF in BMMCs through a mechanism that requires Fyn. (A) Pharmacological characterization of IgE-induced VEGF secretion in MC. Two million BMMCs were pre-incubated with vehicle, Actinomicyn D (Act D; 5 μg/ml), Brefeldin A (BFA; 5μg/ml), Tetanus toxin (TTx; 10 ng/ml) and PP2 (10 μM) for 15 min in BMMC media. Then, cells were stimulated with 1000 ng of IgE for 8 h at 37°C. VEGF in cell supernatants was quantified by ELISA. (B) Role of different Src family kinases on IgE-induced VEGF secretion in MC. Two million BMMCs derived from WT, Fyn −/− and Lyn −/− mice were incubated with different amounts of IgE at 37°C for eight hours. Supernatants were collected and VEGF was determined by ELISA. (C) Time-course of VEGF secretion after the addition of IgE. Two million BMMCs were incubated in cell culture media containing 1000 ng/million cells of IgE at 37°C and supernatants were collected at different times after stimulation. VEGF was determined by ELISA. All results are shown as the mean ± SEM (n = 3-12). *, P < 0.05 compared with basal or time 0, &, P < 0.05 compared with IgE treatment, #, P < 0.05 compared to WT. One way ANOVA post hoc Student-Newman-Keuls (A). Two way ANOVA post hoc Student-Newman-Keuls (B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3750531&req=5

Figure 1: Monomeric IgE induces secretion of VEGF in BMMCs through a mechanism that requires Fyn. (A) Pharmacological characterization of IgE-induced VEGF secretion in MC. Two million BMMCs were pre-incubated with vehicle, Actinomicyn D (Act D; 5 μg/ml), Brefeldin A (BFA; 5μg/ml), Tetanus toxin (TTx; 10 ng/ml) and PP2 (10 μM) for 15 min in BMMC media. Then, cells were stimulated with 1000 ng of IgE for 8 h at 37°C. VEGF in cell supernatants was quantified by ELISA. (B) Role of different Src family kinases on IgE-induced VEGF secretion in MC. Two million BMMCs derived from WT, Fyn −/− and Lyn −/− mice were incubated with different amounts of IgE at 37°C for eight hours. Supernatants were collected and VEGF was determined by ELISA. (C) Time-course of VEGF secretion after the addition of IgE. Two million BMMCs were incubated in cell culture media containing 1000 ng/million cells of IgE at 37°C and supernatants were collected at different times after stimulation. VEGF was determined by ELISA. All results are shown as the mean ± SEM (n = 3-12). *, P < 0.05 compared with basal or time 0, &, P < 0.05 compared with IgE treatment, #, P < 0.05 compared to WT. One way ANOVA post hoc Student-Newman-Keuls (A). Two way ANOVA post hoc Student-Newman-Keuls (B and C).
Mentions: Production of angiogenic factors by MC has been shown to occur few hours after different stimuli, such as hypoxia, antigen or PMA [16,17]. To investigate if monomeric IgE in the absence of any antigen could induce VEGF secretion in this cell type, two million BMMCs were incubated with a monoclonal anti-DNP IgE (1000 ng/ml) for eight hours at 37°C in BMMC media. Supernatants were then collected and the amount of secreted VEGF was determined by ELISA. The addition of IgE to MC induced a significant secretion of VEGF (53.77 ± 2.15 pg/ml in basal conditions vs 117.16 ± 5.45 pg/ml on IgE-stimulated cells; Figure 1A). To gain insight on the mechanism involved in IgE-induced VEGF production, cells were pre-treated with different pharmacological inhibitors 15 minutes before the addition of IgE and secreted VEGF was measured. VEGF secretion was sensitive to actinomycin D (ActD) and brefeldin A (BFA), indicating that de novo transcription and transport from endoplasmic reticulum to the Golgi apparatus [18] was needed for IgE effects to occur. VEGF production was also affected by tetanus toxin (TTx), which inhibits secretion mediated by toxin-sensitive VAMPs (VAMP-1 and −2) [19], and PP2, that inhibits Src family kinases (Figure 1A).

Bottom Line: Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce.In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Farmacobiología, Cinvestav, IPN, Sede Sur, Calzada de los Tenorios 235, Col. Granjas Coapa, Tlalpan CP 14330, Mexico City, Mexico.

ABSTRACT

Background: High concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth.

Methods: For in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE.In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn -/- mice (Wsh Rec Fyn -/-).

Results: Monomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn -/- BMMCs.

Conclusion: Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

Show MeSH
Related in: MedlinePlus