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Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

Taylor AW, Dixit S, Yu J - Int J Ophthalmol Eye Sci (2015)

Bottom Line: The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation.In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation.In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, MA, USA.

ABSTRACT

The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

No MeSH data available.


Related in: MedlinePlus

The conditioned media of RPE eyecups suppresses pHrodo-bioparticle fluorescence in macrophages. The macrophages were cultured as described, and treated with the conditioned media of RPE eye cups (CM) for 24 hoursA) Representative images of the florescence observed with resting, untreated, and CM-treated macrophages.B) The corrected total cellular fluorescence (CTCF) was calculated and made relative to macrophages that were not given beads (Resting). The results are the mean ± SD of 4 cultures analyzing a total of 80 – 100 cells per condition.*Significantly different P ≤ 0.01, and NS is not significant. †P≤ 0.001. The RPE conditioned media suppressed the fluorescence of pHrodo-bioparticles in macrophages indicating suppression of the activation of the phagolysosome.
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Figure 1: The conditioned media of RPE eyecups suppresses pHrodo-bioparticle fluorescence in macrophages. The macrophages were cultured as described, and treated with the conditioned media of RPE eye cups (CM) for 24 hoursA) Representative images of the florescence observed with resting, untreated, and CM-treated macrophages.B) The corrected total cellular fluorescence (CTCF) was calculated and made relative to macrophages that were not given beads (Resting). The results are the mean ± SD of 4 cultures analyzing a total of 80 – 100 cells per condition.*Significantly different P ≤ 0.01, and NS is not significant. †P≤ 0.001. The RPE conditioned media suppressed the fluorescence of pHrodo-bioparticles in macrophages indicating suppression of the activation of the phagolysosome.

Mentions: Previously it was demonstrated that the RPE neuropeptides α-MSH and NPY suppress the activation of the phagolysosome [6]. This was assayed by feeding macrophages, RAW 264.7 cells, with pHrodo-bioparticles that fluoresce red under acidic conditions (Fig. 1A). To see if primary RPE cell monolayers express a soluble factor that suppresses the activation of the phagolysosome in situ eye cups from healthy mouse eyes were cultured for 24 hours and the conditioned media was used to treat the macrophages before given pHrodo-bioparticles. After an additional 24 hours of incubation the cells were assayed for fluorescence. There was a significant suppression in fluorescence of macrophages treated with the eyecup conditioned media (Fig. 1B). Therefore, soluble immunomodulators produced by healthy RPE suppress the activation of the phagolysosome in macrophages.


Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

Taylor AW, Dixit S, Yu J - Int J Ophthalmol Eye Sci (2015)

The conditioned media of RPE eyecups suppresses pHrodo-bioparticle fluorescence in macrophages. The macrophages were cultured as described, and treated with the conditioned media of RPE eye cups (CM) for 24 hoursA) Representative images of the florescence observed with resting, untreated, and CM-treated macrophages.B) The corrected total cellular fluorescence (CTCF) was calculated and made relative to macrophages that were not given beads (Resting). The results are the mean ± SD of 4 cultures analyzing a total of 80 – 100 cells per condition.*Significantly different P ≤ 0.01, and NS is not significant. †P≤ 0.001. The RPE conditioned media suppressed the fluorescence of pHrodo-bioparticles in macrophages indicating suppression of the activation of the phagolysosome.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403791&req=5

Figure 1: The conditioned media of RPE eyecups suppresses pHrodo-bioparticle fluorescence in macrophages. The macrophages were cultured as described, and treated with the conditioned media of RPE eye cups (CM) for 24 hoursA) Representative images of the florescence observed with resting, untreated, and CM-treated macrophages.B) The corrected total cellular fluorescence (CTCF) was calculated and made relative to macrophages that were not given beads (Resting). The results are the mean ± SD of 4 cultures analyzing a total of 80 – 100 cells per condition.*Significantly different P ≤ 0.01, and NS is not significant. †P≤ 0.001. The RPE conditioned media suppressed the fluorescence of pHrodo-bioparticles in macrophages indicating suppression of the activation of the phagolysosome.
Mentions: Previously it was demonstrated that the RPE neuropeptides α-MSH and NPY suppress the activation of the phagolysosome [6]. This was assayed by feeding macrophages, RAW 264.7 cells, with pHrodo-bioparticles that fluoresce red under acidic conditions (Fig. 1A). To see if primary RPE cell monolayers express a soluble factor that suppresses the activation of the phagolysosome in situ eye cups from healthy mouse eyes were cultured for 24 hours and the conditioned media was used to treat the macrophages before given pHrodo-bioparticles. After an additional 24 hours of incubation the cells were assayed for fluorescence. There was a significant suppression in fluorescence of macrophages treated with the eyecup conditioned media (Fig. 1B). Therefore, soluble immunomodulators produced by healthy RPE suppress the activation of the phagolysosome in macrophages.

Bottom Line: The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation.In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation.In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Boston University School of Medicine, Boston, MA, USA.

ABSTRACT

The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye.

No MeSH data available.


Related in: MedlinePlus