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Alpha-melanocyte stimulating hormone (α-MSH) is a post-caspase suppressor of apoptosis in RAW 264.7 macrophages.

Taylor AW - PLoS ONE (2013)

Bottom Line: There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity.In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding.These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA. awtaylor@bu.edu

ABSTRACT
The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

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Effects of α-MSH on apoptotic body uptake and Annexin V binding.A. Untreated macrophages were cultured under the same conditions used for the TUNEL assay in Figure 1. These fixed and TUNEL stained cells were fed to fresh cultures of macrophages treated with α-MSH. The macrophages were assayed 18 hours by flow cytometry for FITC-staining. The mean percent ± SD of cells positive for FITC-staining over background of 4 experiments are presented with significant (P ≤ 0.05) suppression in cells treated with 3 and 10 ng/ml of α-MSH. These results suggest that there is a minor contribution to α-MSH diminution of TUNEL staining associated with α-MSH suppressing the take-up of DNA-fragment containing apoptotic bodies. B. The macrophages were cultured under serum free conditions and assayed by flow cytometry for Annexin V binding. No difference in Annexin V binding was seen between the untreated (SFM) and the α-MSH treated macrophages. The flow cytometry histogram presented is representative of 4 experiments with the same result of no measurable change seen related to α-MSH treatment. These results demonstrate that α-MSH does not suppress the initial steps in the apoptosis pathway.
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pone-0074488-g006: Effects of α-MSH on apoptotic body uptake and Annexin V binding.A. Untreated macrophages were cultured under the same conditions used for the TUNEL assay in Figure 1. These fixed and TUNEL stained cells were fed to fresh cultures of macrophages treated with α-MSH. The macrophages were assayed 18 hours by flow cytometry for FITC-staining. The mean percent ± SD of cells positive for FITC-staining over background of 4 experiments are presented with significant (P ≤ 0.05) suppression in cells treated with 3 and 10 ng/ml of α-MSH. These results suggest that there is a minor contribution to α-MSH diminution of TUNEL staining associated with α-MSH suppressing the take-up of DNA-fragment containing apoptotic bodies. B. The macrophages were cultured under serum free conditions and assayed by flow cytometry for Annexin V binding. No difference in Annexin V binding was seen between the untreated (SFM) and the α-MSH treated macrophages. The flow cytometry histogram presented is representative of 4 experiments with the same result of no measurable change seen related to α-MSH treatment. These results demonstrate that α-MSH does not suppress the initial steps in the apoptosis pathway.

Mentions: Since the RAW cells are phagocytes, there is a possibility that the diminution of TUNEL staining in the α-MSH treated cultures is due reduced uptake of apoptotic bodies with TUNEL detectible DNA fragments. The cells were incubated under serum free conditions, fixed and TUNEL stained. These fixed and stained cells were added to new α-MSH-treated macrophage cultures under serum-free conditions. After incubation the macrophages were washed, assayed by flow cytometry. The analysis gated on intact cells, and the intensity of the TUNEL stain from bound or phagocytized material was measured. There was a significant suppression in the intensity of detectible TUNEL staining in the cells treated with 3 and 10 ng/ml of α-MSH (Figure 6A). This suggests that some of the suppressed TUNEL staining is due to α-MSH inhibiting the phagocytic uptake of apoptotic cells, which would be detected as a reduction in TUNEL staining.


Alpha-melanocyte stimulating hormone (α-MSH) is a post-caspase suppressor of apoptosis in RAW 264.7 macrophages.

Taylor AW - PLoS ONE (2013)

Effects of α-MSH on apoptotic body uptake and Annexin V binding.A. Untreated macrophages were cultured under the same conditions used for the TUNEL assay in Figure 1. These fixed and TUNEL stained cells were fed to fresh cultures of macrophages treated with α-MSH. The macrophages were assayed 18 hours by flow cytometry for FITC-staining. The mean percent ± SD of cells positive for FITC-staining over background of 4 experiments are presented with significant (P ≤ 0.05) suppression in cells treated with 3 and 10 ng/ml of α-MSH. These results suggest that there is a minor contribution to α-MSH diminution of TUNEL staining associated with α-MSH suppressing the take-up of DNA-fragment containing apoptotic bodies. B. The macrophages were cultured under serum free conditions and assayed by flow cytometry for Annexin V binding. No difference in Annexin V binding was seen between the untreated (SFM) and the α-MSH treated macrophages. The flow cytometry histogram presented is representative of 4 experiments with the same result of no measurable change seen related to α-MSH treatment. These results demonstrate that α-MSH does not suppress the initial steps in the apoptosis pathway.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3757010&req=5

pone-0074488-g006: Effects of α-MSH on apoptotic body uptake and Annexin V binding.A. Untreated macrophages were cultured under the same conditions used for the TUNEL assay in Figure 1. These fixed and TUNEL stained cells were fed to fresh cultures of macrophages treated with α-MSH. The macrophages were assayed 18 hours by flow cytometry for FITC-staining. The mean percent ± SD of cells positive for FITC-staining over background of 4 experiments are presented with significant (P ≤ 0.05) suppression in cells treated with 3 and 10 ng/ml of α-MSH. These results suggest that there is a minor contribution to α-MSH diminution of TUNEL staining associated with α-MSH suppressing the take-up of DNA-fragment containing apoptotic bodies. B. The macrophages were cultured under serum free conditions and assayed by flow cytometry for Annexin V binding. No difference in Annexin V binding was seen between the untreated (SFM) and the α-MSH treated macrophages. The flow cytometry histogram presented is representative of 4 experiments with the same result of no measurable change seen related to α-MSH treatment. These results demonstrate that α-MSH does not suppress the initial steps in the apoptosis pathway.
Mentions: Since the RAW cells are phagocytes, there is a possibility that the diminution of TUNEL staining in the α-MSH treated cultures is due reduced uptake of apoptotic bodies with TUNEL detectible DNA fragments. The cells were incubated under serum free conditions, fixed and TUNEL stained. These fixed and stained cells were added to new α-MSH-treated macrophage cultures under serum-free conditions. After incubation the macrophages were washed, assayed by flow cytometry. The analysis gated on intact cells, and the intensity of the TUNEL stain from bound or phagocytized material was measured. There was a significant suppression in the intensity of detectible TUNEL staining in the cells treated with 3 and 10 ng/ml of α-MSH (Figure 6A). This suggests that some of the suppressed TUNEL staining is due to α-MSH inhibiting the phagocytic uptake of apoptotic cells, which would be detected as a reduction in TUNEL staining.

Bottom Line: There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity.In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding.These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, USA. awtaylor@bu.edu

ABSTRACT
The neuropeptide alpha-melanocyte stimulating hormone (α-MSH) is an important regulator of immune cell activity within the immunosuppressive ocular microenvironment. Its constitutive presence not only suppresses macrophage inflammatory activity, it also participates in retinal pigment epithelial cell (RPE) mediated activation of macrophages to function similar to myeloid suppressor cells. In addition, α-MSH promotes survival of the alternatively activated macrophages where without α-MSH RPE induce apoptosis in the macrophages, which is seen as increased TUNEL stained cells. Since there is little know about α-MSH as an anti-apoptotic factor, the effects of α-MSH on caspase activity, mitochondrial membrane potential, Bcl2 to BAX expression, along with TUNEL staining, and Annexin V binding were examined in RAW 264.7 macrophages under serum-starved conditions that trigger apoptosis. There was no effect of α-MSH on activated Caspase 9 and Caspase 3 while there was suppression of Caspase 8 activity. In addition, α-MSH did not improve mitochondrial membrane potential, change the ratio between Bcl-2 and BAX, nor reduce Annexin V binding. These results demonstrate that the diminution in TUNEL staining by α-MSH is through α-MSH mediating suppression of the apoptotic pathway that is post-Caspase 3, but before DNA fragmentation. Therefore, as α-MSH promotes the alternative activation of macrophages it also provides a survival signal, and the potential for the caspases to participate in non-apoptotic activities that can contribute to an immunosuppressive microenvironment.

Show MeSH
Related in: MedlinePlus