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Cytokine-activated endothelial cells delay neutrophil apoptosis in vitro and in vivo. A role for granulocyte/macrophage colony-stimulating factor.

Coxon A, Tang T, Mayadas TN - J. Exp. Med. (1999)

Bottom Line: We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function.The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells.Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The activation of endothelium is important in recruiting neutrophils to sites of inflammation and in modulating their function. We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function. The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells. The in vivo relevance of these findings was investigated in a cytokine-induced model of acute meningitis in mice. Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice. Furthermore, neutrophils recovered from the inflamed cerebrospinal fluid (CSF) exhibited enhanced survival compared with neutrophils isolated from the peripheral blood of the same animals. In unchallenged GM-CSF-deficient mice, the apoptosis of circulating PBNs was similar to wild-type animals; however, after cytokine-induced meningitis, the delay in neutrophil apoptosis typically observed in wild-type mice was attenuated. In contrast, the apoptosis of neutrophils recovered from the CSF of mice of both genotypes was comparable. Taken together, these studies suggest that neutrophil apoptosis is regulated during an inflammatory response, in both intravascular and extravascular compartments. GM-CSF released by activated endothelium can act to increase neutrophil survival and function in the peripheral blood, whereas other factor(s) appear to perform this function in the extravascular space.

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Cytokine-activated endothelial cells significantly delay neutrophil apoptosis, and inhibition of de novo synthesis abrogates expression of the antiapoptotic activity. (A) Neutrophils were allowed to transmigrate through untreated (−) or IL-1β/TNF-α–activated (Cyt.EC) HUVEC monolayers. Buffer alone or buffer plus the chemoattractants FMLP or LTB4 were included in the bottom chamber of the transwell. Neutrophils incubated with untreated HUVECs transmigrated in response to FMLP in the bottom chamber. Control neutrophils were incubated in the presence or absence of the respective chemoattractant, without endothelial cells. (B) Neutrophils were cocultured with HUVEC monolayers for 1 h after HUVECs were stimulated with IL-1β/TNF-α for the indicated times. 2+4 wo indicates that 2 h cytokine–treated HUVECs were washed, incubated for 4 h without cytokines, and then incubated with neutrophils for 1 h. For A and B, *P < 0.005 and #P < 0.05 compared with untreated HUVEC monolayers. (C) Endothelial cells were pretreated with 12 μM actinomycin D (+ Act.D) or 50 μg/ml cycloheximide (+ CX) for 1 h, and IL-1β/TNF-α was added for an additional 1 h. The monolayer was washed, and neutrophils were added for 1 h. #P < 0.05 compared with IL-1β/TNF-α. For A, B, and C, neutrophils were recovered after transmigration or coculture, and apoptosis was assessed 6 h after culturing in fresh medium. Data are expressed as percentage of apoptosis of control neutrophils (% control) to account for the donor variability in the amount of spontaneous apoptosis after 6 h. n = 4 experiments.
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Figure 2: Cytokine-activated endothelial cells significantly delay neutrophil apoptosis, and inhibition of de novo synthesis abrogates expression of the antiapoptotic activity. (A) Neutrophils were allowed to transmigrate through untreated (−) or IL-1β/TNF-α–activated (Cyt.EC) HUVEC monolayers. Buffer alone or buffer plus the chemoattractants FMLP or LTB4 were included in the bottom chamber of the transwell. Neutrophils incubated with untreated HUVECs transmigrated in response to FMLP in the bottom chamber. Control neutrophils were incubated in the presence or absence of the respective chemoattractant, without endothelial cells. (B) Neutrophils were cocultured with HUVEC monolayers for 1 h after HUVECs were stimulated with IL-1β/TNF-α for the indicated times. 2+4 wo indicates that 2 h cytokine–treated HUVECs were washed, incubated for 4 h without cytokines, and then incubated with neutrophils for 1 h. For A and B, *P < 0.005 and #P < 0.05 compared with untreated HUVEC monolayers. (C) Endothelial cells were pretreated with 12 μM actinomycin D (+ Act.D) or 50 μg/ml cycloheximide (+ CX) for 1 h, and IL-1β/TNF-α was added for an additional 1 h. The monolayer was washed, and neutrophils were added for 1 h. #P < 0.05 compared with IL-1β/TNF-α. For A, B, and C, neutrophils were recovered after transmigration or coculture, and apoptosis was assessed 6 h after culturing in fresh medium. Data are expressed as percentage of apoptosis of control neutrophils (% control) to account for the donor variability in the amount of spontaneous apoptosis after 6 h. n = 4 experiments.

Mentions: Endothelial cells are uniquely situated to regulate apoptosis of circulating neutrophils in the blood, as well as those transmigrated into the extravascular space. Therefore, we examined the ability of cytokine-activated endothelial cells to modulate neutrophil apoptosis in an in vitro model of transmigration. Ca2+ and Mg2+ are omitted from the buffers so as to preserve the integrity of the activated endothelial cell monolayer during coculture with neutrophils 2324. Neutrophils were allowed to transmigrate across untreated or IL-1β/TNF-α–treated HUVECs plated in transwells, in the presence or absence of chemoattractants in the bottom chamber. Transmigrated neutrophils were removed, placed in fresh medium, and after 6 h incubation, apoptosis was assessed. Neutrophils that had transmigrated across cytokine-activated endothelial cells had a significant reduction in apoptosis compared with control neutrophils which were similarly treated but were not exposed to endothelial cells (Fig. 2 A). Transmigration across unactivated HUVECs in response to FMLP attenuated apoptosis to a much lesser extent than cytokine-activated HUVECs, suggesting that the endothelial cell–derived antiapoptotic activity is cytokine inducible.


Cytokine-activated endothelial cells delay neutrophil apoptosis in vitro and in vivo. A role for granulocyte/macrophage colony-stimulating factor.

Coxon A, Tang T, Mayadas TN - J. Exp. Med. (1999)

Cytokine-activated endothelial cells significantly delay neutrophil apoptosis, and inhibition of de novo synthesis abrogates expression of the antiapoptotic activity. (A) Neutrophils were allowed to transmigrate through untreated (−) or IL-1β/TNF-α–activated (Cyt.EC) HUVEC monolayers. Buffer alone or buffer plus the chemoattractants FMLP or LTB4 were included in the bottom chamber of the transwell. Neutrophils incubated with untreated HUVECs transmigrated in response to FMLP in the bottom chamber. Control neutrophils were incubated in the presence or absence of the respective chemoattractant, without endothelial cells. (B) Neutrophils were cocultured with HUVEC monolayers for 1 h after HUVECs were stimulated with IL-1β/TNF-α for the indicated times. 2+4 wo indicates that 2 h cytokine–treated HUVECs were washed, incubated for 4 h without cytokines, and then incubated with neutrophils for 1 h. For A and B, *P < 0.005 and #P < 0.05 compared with untreated HUVEC monolayers. (C) Endothelial cells were pretreated with 12 μM actinomycin D (+ Act.D) or 50 μg/ml cycloheximide (+ CX) for 1 h, and IL-1β/TNF-α was added for an additional 1 h. The monolayer was washed, and neutrophils were added for 1 h. #P < 0.05 compared with IL-1β/TNF-α. For A, B, and C, neutrophils were recovered after transmigration or coculture, and apoptosis was assessed 6 h after culturing in fresh medium. Data are expressed as percentage of apoptosis of control neutrophils (% control) to account for the donor variability in the amount of spontaneous apoptosis after 6 h. n = 4 experiments.
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Related In: Results  -  Collection

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Figure 2: Cytokine-activated endothelial cells significantly delay neutrophil apoptosis, and inhibition of de novo synthesis abrogates expression of the antiapoptotic activity. (A) Neutrophils were allowed to transmigrate through untreated (−) or IL-1β/TNF-α–activated (Cyt.EC) HUVEC monolayers. Buffer alone or buffer plus the chemoattractants FMLP or LTB4 were included in the bottom chamber of the transwell. Neutrophils incubated with untreated HUVECs transmigrated in response to FMLP in the bottom chamber. Control neutrophils were incubated in the presence or absence of the respective chemoattractant, without endothelial cells. (B) Neutrophils were cocultured with HUVEC monolayers for 1 h after HUVECs were stimulated with IL-1β/TNF-α for the indicated times. 2+4 wo indicates that 2 h cytokine–treated HUVECs were washed, incubated for 4 h without cytokines, and then incubated with neutrophils for 1 h. For A and B, *P < 0.005 and #P < 0.05 compared with untreated HUVEC monolayers. (C) Endothelial cells were pretreated with 12 μM actinomycin D (+ Act.D) or 50 μg/ml cycloheximide (+ CX) for 1 h, and IL-1β/TNF-α was added for an additional 1 h. The monolayer was washed, and neutrophils were added for 1 h. #P < 0.05 compared with IL-1β/TNF-α. For A, B, and C, neutrophils were recovered after transmigration or coculture, and apoptosis was assessed 6 h after culturing in fresh medium. Data are expressed as percentage of apoptosis of control neutrophils (% control) to account for the donor variability in the amount of spontaneous apoptosis after 6 h. n = 4 experiments.
Mentions: Endothelial cells are uniquely situated to regulate apoptosis of circulating neutrophils in the blood, as well as those transmigrated into the extravascular space. Therefore, we examined the ability of cytokine-activated endothelial cells to modulate neutrophil apoptosis in an in vitro model of transmigration. Ca2+ and Mg2+ are omitted from the buffers so as to preserve the integrity of the activated endothelial cell monolayer during coculture with neutrophils 2324. Neutrophils were allowed to transmigrate across untreated or IL-1β/TNF-α–treated HUVECs plated in transwells, in the presence or absence of chemoattractants in the bottom chamber. Transmigrated neutrophils were removed, placed in fresh medium, and after 6 h incubation, apoptosis was assessed. Neutrophils that had transmigrated across cytokine-activated endothelial cells had a significant reduction in apoptosis compared with control neutrophils which were similarly treated but were not exposed to endothelial cells (Fig. 2 A). Transmigration across unactivated HUVECs in response to FMLP attenuated apoptosis to a much lesser extent than cytokine-activated HUVECs, suggesting that the endothelial cell–derived antiapoptotic activity is cytokine inducible.

Bottom Line: We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function.The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells.Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The activation of endothelium is important in recruiting neutrophils to sites of inflammation and in modulating their function. We demonstrate that conditioned medium from cultured, activated endothelial cells acts to significantly delay the constitutive apoptosis of neutrophils, resulting in their enhanced survival and increased phagocytic function. The antiapoptotic activity is, in part, attributable to granulocyte/macrophage colony-stimulating factor (GM-CSF) secreted by activated endothelial cells. The in vivo relevance of these findings was investigated in a cytokine-induced model of acute meningitis in mice. Peripheral blood neutrophils (PBNs) from mice with meningitis exhibited a delay in apoptosis compared with untreated mice. Furthermore, neutrophils recovered from the inflamed cerebrospinal fluid (CSF) exhibited enhanced survival compared with neutrophils isolated from the peripheral blood of the same animals. In unchallenged GM-CSF-deficient mice, the apoptosis of circulating PBNs was similar to wild-type animals; however, after cytokine-induced meningitis, the delay in neutrophil apoptosis typically observed in wild-type mice was attenuated. In contrast, the apoptosis of neutrophils recovered from the CSF of mice of both genotypes was comparable. Taken together, these studies suggest that neutrophil apoptosis is regulated during an inflammatory response, in both intravascular and extravascular compartments. GM-CSF released by activated endothelium can act to increase neutrophil survival and function in the peripheral blood, whereas other factor(s) appear to perform this function in the extravascular space.

Show MeSH
Related in: MedlinePlus